Mediators of Hedgehog Signaling Pathways, Compositions and Uses Related Thereto

ABSTRACT

The present invention makes available methods and reagents for inhibiting aberrant growth states resulting from hedgehog gain-of-function, ptc loss-of-function or smoothened gain-of-function comprising contacting the cell with a hedgehog antagonist, such as a small molecule, in a sufficient amount to aberrant growth state, e.g., to agonize a normal ptc pathway or antagonize smoothened or hedgehog activity.

This application is a divisional of U.S. Ser. No. 10/484,945, filed Aug.9, 2004, which is a national stage filing under 35 U.S.C. 371 ofInternational Application PCT/US02/24073, filed Jul. 29, 2002, whichclaims priority from U.S. Application 60/308,449, filed Jul. 27, 2001and U.S. Application No. 60/338,031, filed Nov. 13, 2001, thespecifications of each of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Pattern formation is the activity by which embryonic cells form orderedspatial arrangements of differentiated tissues. The physical complexityof higher organisms arises during embryogenesis through the interplay ofcell-intrinsic lineage and cell-extrinsic signaling. Inductiveinteractions are essential to embryonic patterning in vertebratedevelopment from the earliest establishment of the body plan, to thepatterning of the organ systems, to the generation of diverse cell typesduring tissue differentiation (Davidson, E., (1990) Development 108:365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,(1992) Cell 68: 257-270). The effects of developmental cell interactionsare varied. Typically, responding cells are diverted from one route ofcell differentiation to another by inducing cells that differ from boththe uninduced and induced states of the responding cells (inductions).Sometimes cells induce their neighbors to differentiate like themselves(homeogenetic induction); in other cases a cell inhibits its neighborsfrom differentiating like itself. Cell interactions in early developmentmay be sequential, such that an initial induction between two cell typesleads to a progressive amplification of diversity. Moreover, inductiveinteractions occur not only in embryos, but in adult cells as well, andcan act to establish and maintain morphogenetic patterns as well asinduce differentiation (J. B. Gurdon (1992) Cell 68:185-199).

Members of the Hedgehog family of signaling molecules mediate manyimportant short- and long-range patterning processes during invertebrateand vertebrate development. In the fly, a single hedgehog gene regulatessegmental and imaginal disc patterning. In contrast, in vertebrates, ahedgehog gene family is involved in the control of left-right asymmetry,polarity in the CNS, somites and limb, organogenesis, chondrogenesis andspermatogenesis.

The first hedgehog gene was identified by a genetic screen in thefruitfly Drosophila melanogaster (Nilssiein-Volhard, C. and Wieschaus,E. (1980) Nature 287, 795-801). This screen identified a number ofmutations affecting embryonic and larval development. In 1992 and 1993,the molecular nature of the Drosophila hedgehog (hh) gene was reported(C. F., Lee et al. (1992) Cell 71, 33-50), and since then, severalhedgehog homologues have been isolated from various vertebrate species.While only one hedgehog gene has been found in Drosophila and otherinvertebrates, multiple Hedgehog genes are present in vertebrates.

The vertebrate family of hedgehog genes includes at least knit members,e.g., paralogs of the single drosophila hedgehog gene. Exemplaryhedgehog genes and proteins are described in PCT pUblications WO95/18856 and WO 96/17924. Three of these members, herein referred to asDesert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh),apparently exist in all vertebrates, including fish, birds, and mammals.A fourth member, herein referred to as tiggie-winkle hedgehog (Thh),appears specific to fish. Desert hedgehog (Dhh) is expressed principallyin the testes, both in mouse embryonic development and in the adultrodent and human; Indian hedgehog (Ihh) is involved in bone developmentduring embryogenesis and in bone formation in the adult; and, Shh, whichas described above, is primarily involved in morphogenic andneuroinductive activities. Given the critical inductive roles ofhedgehog polypeptides in the development and maintenance of vertebrateorgans, the identification of hedgehog interacting proteins is ofparamount significance in both clinical and research contexts.

The various Hedgehog proteins consist of a signal peptide, a highlyconserved N-terminal region, and a more divergent C-terminal domain. Inaddition to signal sequence cleavage in the secretory pathway (Lee, J.J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) Genes Dev.2635-2645; Chang, D. E. et al. (1994) Development 120; 3339-3353),Hedgehog precursor proteins undergo an internal autoproteolytic cleavagewhich depends on conserved sequences in the C-terminal portion (Lee etal, (1994) Science 266:1528-1537; Porter et al. (1995) Nature374:363-366). This autocleavage leads to a 19 kD N-terminal peptide anda C-terminal peptide of 26-28 kD (Lee et al. (1992) supra; Tabata et al.(1992) supra; Chang et al, (1994) supra; Lee et al. (1994) supra;Bumcrot, D. A., et al. (1995) Mol. Cell. Biol. 15:2294-2303; Porter etal. (1995) supra; Ekker, S. C. et al. (1995) Curr. Biol. 5:944-955; Lai,C. J. et al. (1995) Development 121:2349-2360). The N-terminal peptidestays tightly associated with the surface of cells in which it wassynthesized, while the C-terminal peptide is freely diffusible both invitro and in vivo (Porter et al. (1995) Nature 374:363; Lee et al.(1994) supra; Bumcrot et al. (1995) supra; Mart', E. et al. (1995)Development 121:2537-2547; Roelink, H. et al. (1995) Cell 81:445-455).Interestingly, cell surface retention of the N-terminal peptide isdependent on autocleavage, as a truncated form of HH encoded by an RNAwhich terminates precisely at the normal position of internal cleavageis diffusible in vitro (Porter et al. (1995) supra) and in vivo (Porter,J. A. et al. (1996) Cell 86, 21-34). Biochemical studies have shown thatthe autoproteolytic cleavage of the HH precursor protein proceedsthrough an internal thioester intermediate that subsequently is cleavedin a nucleophilic substitution. It is likely that the nucleophile is asmall lipophilic molecule that becomes covalently bound to theC-terminal end of the N-peptide (Porter et al. (1996) supra), tetheringit to the cell surface. The biological implications are profound. As aresult of the tethering, a high local concentration of N-terminalHedgehog peptide is generated on the surface of the Hedgehog producingcells. It is this N-terminal peptide which is both necessary andsufficient for short- and long-range Hedgehog signaling activities inDrosophila and vertebrates (Porter et al. (1995) supra; Ekker et al.(1995) supra; Lai et al. (1995) supra; Roelink, H. et al. (1995) Cell81:445-455; Porter et al, (1996) supra; Fietz, M. J. et al. (1995) Curr.Biol. 5:643-651; Fan, C.-M. et al. (1995) Cell 81:457-465; Mart', E., etal. (1995) Nature 375:322-325; Lopez-Martinez et al. (1995) Curr. Biol5:791-795; Ekker, S. C. et al. (1995) Development 121:2337-2347; Forbes,A. J. et al. (1996) Development 122:1125-1135).

HH has been implicated in short- and long-range patterning processes atvarious sites during Drosophila development. In the establishment ofsegment polarity in early embryos, it has short-range effects whichappear to be directly mediated, while in the patterning of the imaginaldiscs, it induces long range effects via the induction of secondarysignals.

In vertebrates, several hedgehog genes have been cloned in the past fewyears. Of these genes, Shh has received most of the experimentalattention, as it is expressed in different organizing centers that arethe sources of signals that pattern neighboring tissues. Recent evidenceindicates that Shh is involved in these interactions.

The expression of Shh starts shortly after the onset of gastrulation inthe presumptive midline mesoderm, the node in the mouse (Chang et al.(1994) supra.; Echelard, Y. et al. (1993) Cell 75; 1417-1430), the rat(Roelink, H. et al. (1994) Cell 76:761-775) and the chick (Riddle, R. D.et al. (1993) Cell 75:1401-1416), and the shield in the zebrafish (Ekkeret al, (1995) supra; Krauss, S, et al. (1993) Cell 75; 1431-1444). Inchick embyros, the Shh expression pattern in the node develops aleft-right asymmetry, which appears to be responsible for the left-rightsitus of the heart (Levin, M. et al. (1995) Cell 82:803-814).

In the CNS, Shh from the notochord and the floorplate appears to induceventral cell fates. When ectopically expressed, Shh leads to aventralization of large regions of the mid- and hindbrain in mouse(Echelard et al. (1993) supra; Goodrich, L. V. et al. (1996) Genes Dev,10:301-312), Xenopus (Roelink, H. et al. (1994) supra; Ruiz i Altaba, etal. (1995) Mol. Cell. Neurosci. 6:106-121), and zebrafish (Ekker et al,(1995) supra; Krauss et al. (1993) supra; Hammerschmidt, M., et al.(1996) Genes Dev. 10:647-658). In explants of intermediate neuroectodermat spinal cord levels, Shh protein induces floorplate and motor neurondevelopment with distinct, concentration thresholds, floor plate at highand motor neurons at lower concentrations (Roelink et al. (1995) supra;Mart' et al. (1995) supra; Tanahe, Y. et al. (1995) Curr. Biol,5:651-658). Moreover, antibody blocking suggests that Shh produced bythe notochord is required for notochord-mediated induction of motorneuron fates (Mart' et al. (1995) supra). Thus, high concentration ofShh on the surface of Shh-producing midline cells appears to account forthe contact-mediated induction of floorplate observed in vitro (Placzek,M. et al. (1993) Development 117:205-218), and the midline positioningof the floorplate immediately above the notochord in vivo. Lowerconcentrations of Shh released from the notochord and the floorplatepresumably induce motor neurons at more distant ventrolateral regions ina process that has been shown to be contact-independent in vitro(Yarnada, T. et al. (1993) Cell 73:673-686). In explants taken atmidbrain and forebrain levels, Shh also induces the appropriateventrolateral neuronal cell types, dopaminergic (Heynes, M. et al.(1995) Neuron 15:35.44; Wang, M. Z. et al. (1995) Nature Med.1:1184-1188) and cholinergic (Ericson, J. et al. (1995) Cell 81:747-756)precursors, respectively, indicating that Shh is a common inducer ofventral specification over the entire length of the CNS. Theseobservations raise a question as to how the differential response to Shhis regulated at particular anteroposterior positions.

Shh from the midline also patterns the paraxial regions of thevertebrate embryo, the somites in the trunk (Fan et al. (1995) supra)and the head mesenchyme rostral of the somites (Hammerschmidt et al.(1996) supra). In chick and mouse paraxial mesoderm explants, Shhpromotes the expression of sclerotome specific markers like Payl andTwist, at the expense of the dermamyotomal marker Pax3. Moreover, filterbarrier experiments suggest that Shh mediates the induction of thesclerotome directly rather than by activation of a secondary signalingmechanism (Fan, C.-M. and Tessier-Lavigne, M. (1994) Cell 79,1175-1186).

Shh also induces myotomal gene expression (Harnmerschrnidt et al. (1996)supra; Johnson, R. L. et al. (1994) Cell 79:1165-1173; MUnsterberg, A.E, et al. (1995) Genes Dev, 9:2911-2922; Weinberg, E. S. et al. (1996)Development 122:271-280), although recent experiments indicate thatmembers of the WNT family, vertebrate homologues of Drosophila wingless,are required in concert (Münsterberg et al. (1995) supra). Puzzlingly,myotomal induction in chicks requires higher Shh concentrations than theinduction of sclerotomal markers (Mansterberg et al. (1995) supra),although the sclerotome originates from somitic, cells positioned muchcloser to the notochord. Similar results were obtained in the zebrafish,where high concentrations of Hedgehog induce myotoinal and represssclerotomal marker gene expression (Hammerschmidt el al. (1996) supra).In contrast to anmiotes, however, these observations are consistent withthe architecture of the fish embryo, as here, the myotome is thepredominant and more axial component of the somites. Thus, modulation ofShh signaling and the acquisition of new signaling factors may havemodified the somite structure during vertebrate evolution.

In the vertebrate limb buds, a subset of posterior mesenchymal cells,the “Zone of polarizing activity” (ZPA), regulates anteroposterior digitidentity (reviewed in Honig, L. S. (1981) Nature 291:72-73). Ectopicexpression of Shh or application of beads soaked in Shh peptide mimicsthe effect of anterior ZPA grafts, generating a mirror image duplicationof digits (Chang et al. (1994) supra; Lopez-Martinez et al. (1995)supra; Riddle et al. (1993) svra) (FIG. 2 g). Thus, digit identityappears to depend primarily on Shh concentration, although it ispossible that other signals may relay this information over thesubstantial distances that appear to be required for AP patterning(100-150 μm). Similar to the interaction of HH and DPP in the Drosophilaimaginal discs, Shh in the vertebrate limb bud activates the expressionof Bmp 2 (Francis, et al., (1994) Development 120:2119-218), a dpphomologue. However, unlike DPP in Drosophila, Bmp2 fails to mimic thepolarizing effect of Shh upon ectopic application in the chick limb bud(Francis et al. (1994) supra). In addition to anteroposteriorpatterning, Shh also appears to be involved in the regulation of theproximodistal outgrowth of the limbs by inducing the synthesis of thefibroblast growth factor FGF4 in the posterior apical ectodermal ridge(Laufer, E. et al. (1994) Cell 79:993-1003; Niswander, et al. (1994)Nature 371:609-612).

The close relationship between Hedgehog proteins and BMPs is likely tohave been conserved at many, but probably not all sites of vertebrateHedgehog expression. For example, in the chick hindgut, Shh has beenshown to induce the expression of Bmp4, another vertebrate dpp homologue(Roberts, D. J. et al. (1995) Development 121:3163-3174). Furthermore,Shh and Bmp2, 4, or 6 show a striking correlation in their expression inepithelial and mesenchymal cells of the stomach, the urogenital system,the lung, the tooth buds and the hair follicles (Bitgood, M. J. andMcMahon, A. P. (1995) Dcv. Biol, 172:126-138). Further, Ihh, one of thetwo other mouse Hedgehog genes, is expressed adjacent to MIT expressingcells in the gut and developing cartilage (Bitgood and McMahon (1995)supra).

Recent evidence suggests a model in which ihh plays a crucial role inthe regulation of chondrogenic development (Roberts et al. (1995)supra). During cartilage formation, chondrocytes proceed from aproliferating state via an intermediate, prehypertrophic state todifferentiated hypertrophic chondrocytes. Ihh is expressed in theprehypertrophic chondrocytes and initiates a signaling cascade thatleads to the blockage of chondrocyte differentiation. Its direct targetis the perichondrium around the Ihh expression domain, which responds bythe expression of Gil and Patched (Ptc), conserved transcriptionaltargets of Hedgehog signals (see below). Most likely, this leads tosecondary signaling resulting in the synthesis of parathyroidhormone-related protein (PTHrP) in the periarticular perichondrium.PTHrP itself signals back to the prehypertrophic chondrocytes blockingtheir further differentiation. At the same time, PTHrP repressesexpression of Ihh, thereby forming a negative feedback loop thatmodulates the rate of chondrocyte differentiation.

Patched was originally identified in Drosophila as a segment polaritygene, one of a group of developmental genes that affect celldifferentiation within the individual segments that occur in ahomologous series along the anterior-posterior axis of the embryo. SeeHooper, J. E. et al. (1989) Cell 59:751; and Nakano, Y, et at, (1989)Nature 341:508. Patterns of expression of the vertebrate homologue ofpatched suggest its involvement in the development of neural tube,skeleton, limbs, craniofacial structure, and skin.

Genetic and functional studies demonstrate that patched is part of thehedgehog signaling cascade, an evolutionarily conserved pathway thatregulates expression of a number of downstream genes. See Perrimon, N.(1995) Cell 80:517; and Perrimon, N. (1996) Cell 86:513. Patchedparticipates in the constitutive transcriptional repression of thetarget genes; its effect is opposed by a secreted glycoprotein, encodedby hedgehog, or a vertebrate homologue, which induces transcriptionalactivation. Genes under control of this pathway include members of theWnt and TGF-beta families.

Patched proteins possess two large extracellular domains, twelvetransmembrane segments, and several cytoplasmic segments. See Hooper,supra; Nakano, supra; Johnson, R. L. et al. (1996) Science 272:1668; andHahn, H. et al, (1996) Cell 85:841. The biochemical role of patched inthe hedgehog signaling pathway is unclear. Direct interaction with thehedgehog protein has, however, been reported (Chen, V. et al. (1996)Cell 87:553), and patched may participate in a hedgehog receptor complexalong with another transmembrane protein encoded by the smoothened gene.See Perrimon, supra; and Chen, supra.

The human homologue of patched was recently cloned and mapped toChromosome 9q22.3. See Johnson, supra; and Hahn, supra. This region hasbeen implicated in basal cell nevus syndrome (BCNS), which ischaracterized by developmental abnormalities including rib andcraniofacial alterations, abnormalities of the hands and feet, and spinabifida.

BCNS also predisposes to multiple tumor types, the most frequent beingbasal cell carcinomas (BCC) that occur in many locations on the body andappear within the first two decades of life. Most cases of BCC, however,are unrelated to the syndrome and arise sporadically in small numbers onsun-exposed sites of middle-aged or older people of northern Europeanancestry.

Recent studies in BCNS-related and sporadic BCC suggest that afunctional loss of both alleles of patched leads to development of BCC.See Johnson, supra; Hahn, supra; and Gailani, M. R. et al. (1996) NatureGenetics 14:78. Single allele deletions of chromosome 9q22.3 occurfrequently in both sporadic and hereditary BCC. Linkage analysisrevealed that the defective inherited allele was retained and the normalallele was lost in tumors from BCNS patients.

Sporadic tumors also demonstrated a loss of both functional alleles ofpatched. Of twelve tumors in which patched mutations were identifiedwith a single strand conformational polymorphism screening assay, ninehad chromosomal deletion of the second allele and the other three hadinactivating mutations in both alleles (Gailani, supra). The alterationsdid not occur in the corresponding germline DNA.

Most of the identified mutations resulted in premature stop codons orframe shifts. Lench, N. J., et al., Hum. Genet. 1997 October; 100(5-6):497-502. Several, however, were point mutations leading to amino acidsubstitutions in either extracellular or cytoplasmic domains. Thesesites of mutation may indicate functional importance for interactionwith extracellular proteins or with cytoplasmic members of thedownstream signaling pathway.

The involvement of patched in the inhibition of gene expression and theoccurrence of frequent allelic deletions of patched in BCC support atumor suppressor function for this gene. Its role in the regulation ofgene families known to be involved in cell signaling and intercellularcommunication provides a possible mechanism of tumor suppression.

SUMMARY OF THE INVENTION

The present invention makes available methods and reagents forinhibiting activation of the hedgehog signaling pathway, e.g., toinhibit aberrant growth states resulting from phenotypes such as ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function, comprising contacting the cell with an agent, such asa small molecule, in a sufficient amount to agonize a normal ptcactivity, antagonize a normal hedgehog activity, or antagonizesmoothened activity, e.g., to reverse or control the aberrant growthstate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-31 depict reactions useful for synthesizing compounds accordingto the present invention.

FIG. 32 a-j illustrates representative compounds according to thepresent invention.

FIG. 33 shows the effect of compounds of the invention on hedgehogsignalling in human basal cell carcinomas (BCCs).

FIG. 34 illustrates the effect of compounds of the invention onapoptosis in BCC tissue.

FIG. 35 portrays the effect of compounds of the invention on hedgehogsignalling in S-12 and HT-29 cells.

FIG. 36 depicts the effect of compounds of the invention on hedgehogsignalling in PC-3 and RT-4

FIG. 37 provides sequences of probes and primers used to evaluatehedgehog activity in cells.

FIGS. 38 and 40 illustrate the differential effects of compounds of theinvention on GLI-1 and GAPDH expression in pct-null cells.

FIGS. 39 and 41 show the differential effects of compounds of theinvention on GLI-1 and GAPDH expression in HEPM cells.

BEST MODES FOR CARRYING OUT THE INVENTION Detailed Description of theInvention I. Overview

The present invention relates to the discovery that signal transductionpathways regulated by hedgehog, patched (ptc), gli and/or smoothened canbe inhibited, at least in part, by small molecules. While not wishing tobe bound by any particular theory, the activation of a receptor may bethe mechanism by which these agents act. For example, the ability ofthese agents to inhibit proliferation of patched loss-of-function(ptc^(lof)) cells may be due to the ability of such molecules tointeract with hedgehog, patched, or smoothened, or at least to interferewith the ability of those proteins to activate a hedgehog, ptc, and/orsmoothened-mediated signal transduction pathway.

It is, therefore, specifically contemplated that these small moleculeswhich intefere with aspects of hedgehog, ptc, or smoothened signaltransduction activity will likewise be capable of inhibitingproliferation (or other biological consequences) in normal cells and/orcells having a patched loss-of-function phenotype, a hedgehoggain-of-function phenotype, or a smoothened gain-of-function phenotype.Thus, it is contemplated that in certain embodiments, these compoundsmay be useful for inhibiting hedgehog activity in normal cells, e.g.,which do not have a genetic mutation that activates the hedgehogpathway. In preferred embodiments, the subject inhibitors are organicmolecules having a molecular weight less than 2500 amu, more preferablyless than 1500 amu, and even more preferably less than 750 amu, and arecapable of inhibiting at least some of the biological activities ofhedgehog proteins, preferably specifically in target cells.

Thus, the methods of the present invention include the use of smallmolecules which agonize ptc inhibition of hedgehog signalling, such asby inhibiting activation of smoothened or downstream components of thesignal pathway, in the regulation of repair and/or functionalperformance of a wide range of cells, tissues and organs, includingnormal cells, tissues, and organs, as well as those having the phenotypeof ptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function. For instance, the subject method has therapeutic andcosmetic applications ranging from regulation of neural tissues, boneand cartilage formation and repair, regulation of spermatogenesis,regulation of smooth muscle, regulation of lung, liver and other organsarising from the primative gut, regulation of hematopoietic function,regulation of skin and hair growth, etc. Moreover, the subject methodscan be performed on cells that are provided in culture (in vitro), or oncells in a whole animal (in vivo). See, for example, PCT publications WO95/18856 and WO 96/17924 (the specifications of which are expresslyincorporated by reference herein).

In a preferred embodiment, the subject method can be to treat epithelialcells having a phenotype of ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function. For instance, thesubject method can be used in treating or preventing basal cellcarcinoma or other hedgehog pathway-related disorders.

In certain embodiments, a subject antagonist may inhibit activation of ahedgehog pathway by binding to smoothened. In certain embodiments, asubject antagonist may inhibit activation of a hedgehog pathway bybinding to patched.

In another preferred embodiment, the subject method can be used as partof a treatment regimen for malignant medulloblastoma and other primaryCNS malignant neuroectodermal tumors.

In another aspect, the present invention provides pharmaceuticalpreparations comprising, as an active ingredient, a hedgehog antagonist,ptc agonist, or smoothened antagonist such as described herein,formulated in an amount sufficient to inhibit, in vivo, proliferation orother biological consequences of ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function.

The subject treatments using hedgehog antagonists, patched agonists, orsmoothened antagonists can be effective for both human and animalsubjects. Animal subjects to which the invention is applicable extend toboth domestic animals and livestock, raised either as pets or forcommercial purposes. Examples are dogs, cats, cattle, horses, sheep,hogs, and goats.

II. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The phrase “aberrant modification or mutation” of a gene refers to suchgenetic lesions as, for example, deletions, substitution or addition ofnucleotides to a gene, as well as gross chromosomal rearrangements ofthe gene and/or abnormal methylation of the gene. Likewise,mis-expression of a gene refers to aberrant levels of transcription ofthe gene relative to those levels in a normal cell under similarconditions, as well as non-wild-type splicing of mRNA transcribed fromthe gene.

“Basal cell carcinomas” exist in a variety of clinical and histologicalforms such as nodular-ulcerative, superficial, pigmented, morphealike,fibroepithelioma and nevoid syndrome. Basal cell carcinomas are the mostcommon cutaneous neoplasms found in humans. The majority of new cases ofnonmelanoma skin cancers fall into this category.

“Burn wounds” refer to cases where large surface areas of skin have beenremoved or lost from an individual due to heat and/or chemical agents.

The term “carcinoma.” refers to a malignant new growth made up ofepithelial cells tending to infiltrate surrounding tissues and to giverise to metastases. Exemplary carcinomas include: “basal cellcarcinoma”, which is an epithelial tumor of the skin that, while seldommetastasizing, has potentialities for local invasion and destruction;“squamous cell carcinoma”, which refers to carcinomas arising fromsquamous epithelium and having cuboid cells; “carcinosarcoma”, whichinclude malignant tumors composed of carcinomatous and sarcomatoustissues; “adenocystic carcinoma”, carcinoma marked by cylinders or bandsof hyaline or mucinous stroma separated or surrounded by nests or cordsof small epithelial cells, occurring in the mammary and salivary glands,and mucous glands of the respiratory tract; “epidermoid carcinoma”,which refers to cancerous cells which tend to differentiate in the sameway as those of the epidermis; i.e., they tend to form prickle cells andundergo cornification; “nasopharyngeal carcinoma”, which refers to amalignant tumor arising in the epithelial lining of the space behind thenose; and “renal cell carcinoma”, which pertains to carcinoma of therenal parenchyma composed of tubular cells in varying arrangements.Other carcinomatous epithelial growths are “papillomas”, which refers tobenign tumors derived from epithelium and having a papillomavirus as acausative agent; and “epidermoidomas”, which refers to a cerebral ormeningeal tumor formed by inclusion of ectodermal elements at the timeof closure of the neural groove.

The “corium” or “dermis” refers to the layer of the skin deep to theepidermis, consisting of a dense bed of vascular connective tissue, andcontaining the nerves and terminal organs of sensation. The hair roots,and sebaceous and sweat glands are structures of the epidermis which aredeeply embedded in the dermis.

“Dental tissue” refers to tissue in the mouth that is similar toepithelial tissue, for example gum tissue. The method of the presentinvention is useful for treating periodontal disease.

“Dermal skin ulcers” refer to lesions on the skin caused by superficialloss of tissue, usually with inflammation. Dermal skin ulcers that canbe treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect.

An “effective amount” of, e.g., a hedgehog antagonist, with respect tothe subject method of treatment, refers to an amount of the antagonistin a preparation which, when applied as part of a desired dosage regimenbrings about, e.g. a change in the rate of cell proliferation and/or thestate of differentiation of a cell and/or rate of survival of a cellaccording to clinically acceptable standards for the disorder to betreated or the cosmetic purpose.

The terms “epithelia”, “epithelial” and “epithelium” refer to thecellular covering of internal and external body surfaces (cutaneous,mucous and serous), including the glands and other structures derivedtherefrom, e.g., corneal, esophageal, epidermal, and hair follicleepithelial cells. Other exemplary epithlelial tissue includes: olfactoryepithelium, which is the pseudostratitled epithelium lining theolfactory region of the nasal cavity, and containing the receptors forthe sense of smell; glandular epithelium, which refers to epitheliumcomposed of secreting cells; squamous epithelium, which refers toepithelium composed of flattened plate-like cells. The term epitheliumcan also refer to transitional epithelium, like that which ischaracteristically found lining hollow organs that are subject to greatmechanical change due to contraction and distention, e.g., tissue whichrepresents a transition between stratified squamous and columnarepithelium.

The term “epithelialization” refers to healing by the growth ofepithelial tissue over a denuded surface.

The term “epidermal gland” refers to an aggregation of cells associatedwith the epidermis and specialized to secrete or excrete materials notrelated to their ordinary metabolic needs. For example, “sebaceousglands” are holocrine glands in the corium that secrete an oilysubstance and sebum. The term “sweat glands” refers to glands thatsecrete sweat, situated in the corium or subcutaneous tissue, opening bya duct on the body surface.

The term “epidermis” refers to the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers: basal layer composed of columnar cellsarranged perpendicularly; prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, cornifiednon-nucleated cells, in the epidermis of the general body surface, theclear layer is usually absent.

“Excisional wounds” include tears, abrasions, cuts, punctures orlacerations in the epithelial layer of the skin and may extend into thedermal layer and even into subcutaneous fat and beyond. Excisionalwounds can result from surgical procedures or from accidentalpenetration of the skin.

The “growth state” of a cell refers to the rate of proliferation of thecell and/or the state of differentiation of the cell. An “altered growthstate” is a growth state characterized by an abnormal rate ofproliferation, e.g., a cell exhibiting an increased or decreased rate ofproliferation relative to a normal cell.

The term “hair” refers to a threadlike structure, especially thespecialized epidermal structure composed of keratin and developing froma papilla sunk in the corium, produced only by mammals andcharacteristic of that group of animals. Also, “hair” may refer to theaggregate of such hairs. A “hair follicle” refers to one of thetubular-invaginations of the epidermis enclosing the hairs, and fromwhich the hairs grow. “Hair follicle epithelial cells” refers toepithelial cells that surround the dermal papilla in the hair follicle,e.g., stem cells, outer root sheath cells, matrix cells, and inner rootsheath cells. Such cells may be normal non-malignant cells, ortransformed/immortalized cells.

The term “hedgehog antagonist” refers to an agent that potentiates orrecapitulates the bioactivity of patched, such as to represstranscription of target genes. Preferred hedgehog antagonists can beused to overcome a ptc loss-of function and/or a smoothenedgain-of-function, the latter also being referred to as smoothenedantagonists. The term ‘hedgehog antagonist’ as used herein refers notonly to any agent that may act by directly inhibiting the normalfunction of the hedgehog protein, but also to any agent that inhibitsthe hedgehog signalling pathway, and thus recapitulates the function ofptc.

The term “hedgehog gain-of-function” refers to an aberrant modificationor mutation of a ptc gene, hedgehog gene, or smoothened gene, or adecrease (or loss) in the level of expression of such a gene, whichresults in a phenotype which resembles contacting a cell with a hedgehogprotein, e.g., aberrant activation of a hedgehog pathway. Thegain-of-function may include a loss of the ability of the ptc geneproduct to regulate the level of expression of Ci genes, e.g., Gli1,Gli2, and Gli3. The term ‘hedgehog gain-of-function’ is also used hereinto refer to any similar cellular phenotype (e.g., exhibiting excessproliferation) that occurs due to an alteration anywhere in the hedgehogsignal transduction pathway, including, but not limited to, amodification or mutation of hedgehog itself. For example, a tumor cellwith an abnormally high proliferation rate due to activation of thehedgehog signalling pathway would have a ‘hedgehog gain-of-function’phenotype, even if hedgehog is not mutated in that cell.

As used herein, “immortalized cells” refers to cells that have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

“Internal epithelial tissue” refers to tissue inside the body that hascharacteristics similar to the epidermal layer in the skin. Examplesinclude the lining of the intestine. The method of the present inventionis useful for promoting the healing of certain internal wounds, forexample wounds resulting from surgery.

The term “keratosis” refers to proliferative skin disorder characterizedby hyperplasia of the horny layer of the epidermis. Exemplary keratoticdisorders include keratosis follicularis, keratosis palmaris etplantaris, keratosis pharyngea, keratosis pilaris, and actinickeratosis.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

The term “nail” refers to the horny cutaneous plate on the dorsalsurface of the distal end of a finger or toe.

The term “patched loss-of-function” refers to an aberrant modificationor mutation of a ptc, gene, or a decreased level of expression of thegene, which results in a phenotype which resembles contacting a cellwith a hedgehog protein, e.g., aberrant activation of a hedgehogpathway. The loss-of-function may include a loss of the ability of theptc gene product to regulate the level of expression of Ci genes, e.g.,Gil1, Gli2 and Gli3. The term ‘ptc loss-of-function’ is also used hereinto refer to any similar cellular phenotype (e.g., exhibiting excessproliferation) that occurs due to an alteration anywhere in the hedgehogsignal transduction pathway, including, but not limited to, amodification or mutation ofptc itself. For example, a tumor cell with anabnormally high proliferation rate due to activation of the hedgehogsignalling pathway would have a ‘ptc loss-of-function’ phenotype, evenif ptc is not mutated in that cell.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The term “prodrug” is intended to encompass compounds that, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties that are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

Throughout this application, the term “proliferative skin disorder”refers to any disease/disorder of the skin marked by unwanted oraberrant proliferation of cutaneous tissue. These conditions aretypically characterized by epidermal cell proliferation or incompletecell differentiation, and include, for example, X-linked ichthyosis,psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytichyperkeratosis, and seborrheic dermatitis. For example,epidermodysplasia is a form of faulty development of the epidermis.Another example is “epidermolysis”, which refers to a loosened state ofthe epidermis with formation of blebs and bullae either spontaneously orat the site of trauma.

As used herein, the term “psoriasis” refers to a hyperproliferative skindisorder that alters the skids regulatory mechanisms. In particular,lesions are formed which involve primary and secondary alterations inepidermal proliferation, inflammatory responses of the skin, and anexpression of regulatory molecules such as lymphokines and inflammatoryfactors. Psoriatic skin is morphologically characterized by an increasedturnover of epidermal cells, thickened epidermis, abnormalkeratinization, inflammatory cell infiltrates into the dermis layer andpolymorphonuclear leukocyte infiltration into the epidermis layerresulting in an increase in the basal cell cycle. Additionally,hyperkeratotic and parakeratotic cells are present.

The term “skin” refers to the outer protective covering of the body,consisting of the corium and the epidermis, and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” may beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used.

The term “smoothened gain-of-function” refers to an aberrantmodification or mutation of a sire gene, or an increased level ofexpression of the gene, which results in a phenotype that resemblescontacting a cell with a hedgehog protein, e.g., aberrant activation ofa hedgehog pathway. While not wishing to be hound by any particulartheory, it is noted that ptc may not signal directly into the cell, butrather interact with smoothened, another membrane-bound protein locateddownstream of pie in hedgehog signaling (Mango et al., (1996) Nature384: 177-179). The gene smo is a segment-polarity gene required for thecorrect patterning of every segment in Drosophila (Alcedo et al., (1996)Cell 86: 221-232). Human homologs of smo have been identified. See, forexample, Stone et al. (1996) Nature 384:129-134, and GenBank accessionU84401. The smoothened gene encodes an integral membrane protein withcharacteristics of heterotrimeric G-protein-coupled receptors; i.e., 7transmembrane regions. This protein shows homology to the DrosophilaFrizzled (Fz) protein, a member of the wingless pathway, it wasoriginally thought that smo encodes a receptor of the Hh signal.However, this suggestion was subsequently disproved, as evidence forgebeing the Hh receptor was obtained. Cells that express Smo fail to bindHh, indicating that smo does not interact directly with Hal (Masse,(1996) Nature 384: 119-120). Rather, the binding of Sonic hedgehog (SHU)to its receptor, PTCH, is thought to prevent normal inhibition by PTCHof smoothened (SMO), a seven-span transmembrane protein.

Recently, it has been reported that activating smoothened mutationsoccur in sporadic basal cell carcinoma, Xie et al, (1998) Nature 391:90-2, and primitive neuroectodermal tumors of the central nervoussystem, Reifenberger et al. (1998) cancer Res 58: 1798-803.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

As used herein, “transformed cells” refers to cells that havespontaneously converted to a state of unrestrained growth, i.e, theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where in and R₈ are as definedabove.

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkenyl group.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulthydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkylryl, and —S—(C₁₋₁₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In certain such embodiments, neither R₉ and R₁₀ is attached toN by a carbonyl, e.g., the amine is not an amide or imide, and the amineis preferably basic, e.g., its conjugate acid has a pK_(a) above 7. Ineven more preferred embodiments, R₉ and R₁₀ (and optionally R′₁₀) eachindependently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include that may be unstable.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, stilfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromaticor heteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and or heterocyclyls.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and representsa hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or a pharmaceuticallyacceptable salt, R′₁₁ represents a hydrogen, an alkyl, an alkenyl or—(CH₂)_(m)—R₈, where m and R₈ are as defined above. Where X is an oxygenand R₁₁ or R′₁₁ is not hydrogen, the formula represents an “ester”.Where X is an oxygen, and R₁₁ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R₁₁ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen, and R′₁₁ is hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate,” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The terms “heterocycyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3 to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,plithalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, anaromatic or heteroaromatic moiety, —CN, or the like.

As used herein, the term “nitro” means —NO₂, the term “halogen”designates F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂.

A “phosphonamidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, Q₂ represents O, S or N, andR₄₈ represents a lower alkyl or an aryl, Q₂ represents O, S or N.

A “phosphoramidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S or N.

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, for example, an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheteocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W. Wuts, P.G.M.Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₈, m and R₈ being definedabove.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above,

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The terms trityl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof and other mixtures thereof, as falling within the scopeof the invention. Additional asymmetric carbon atoms may be present in asubstituent such as an alkyl group. All such isomers, as well asmixtures thereof are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts may be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit hedgehogsignaling), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds of the present invention may be prepared by themethods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

III. Exemplary Compounds of the Invention.

As described in further detail below, it is contemplated that thesubject methods can be carried out using a variety of different smallmolecules which can be readily identified, for example, by such drugscreening assays as described herein. For example, compounds useful inthe subject methods include compounds may be represented by generalformula (I):

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—,—(R₇)N—N(R₇)—-ON(R₇)—, or a direct bond, preferably —N(R₇)—, —O—, —S—,or a direct bond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring.

Ar represents a substituted or unsubstituted aryl or heteroaryl ring,such as a substituted or unsubstituted phenyl ring;

R₁ represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl,aryl, heteroaryl, heterocyclyl, or cycloalkyl, including polycyclicgroups;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably front 0-4) units selected from CK₂, NK, O, and S, wherein Krepresents, independently for each occurrence, H or lower alkyl.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X—Y—Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes an aryl or heteroaryl ring,optionally substituted with from 1-5 substituents, such as nitro,halogen, cyano, lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy,alkylamino, a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to the aryl or heteroaryl ring.

In certain embodiments, X and the ring comprising A are disposed on Arin a meta (i.e., 1,3) relationship.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, the subject compound is selected from thecompounds depicted in FIG. 32.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (II):

wherein, as valence and stability, permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₁₇)—, or a direct bond, preferably —N(R₇)—, —S—, or a direct bond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, mil, or heteroaryl ring fusedto the ring to Which it is attached, preferably an aryl or heteroarylring.

R₁ represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl,aryl, heteroaryl, heterocyclyl, or cycloalkyl, including polycyclicgroups;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic, structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₃ represents from 0-4 substituents on the ring to Which it is attached,such as halogen, hydroxyl, alkoxy, amino, alkylamino, cyano, nitro,substituted or unsubstituted lower alkyl, and acyl, preferably halogen,lower alkoxy, or substituted or unsubstituted lower alkyl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain haying from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S, wherein Krepresents, independently fix each occurrence, H or lower alkyl.

In certain embodiments, at least one of Z and X is not a direct bond, incertain embodiments, X—Y—Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes an aryl or heteroaryl ring,optionally substituted with from 1-5 substituents, such as nitro,halogen, cyano, lower alkyl, acylamino (e.g., R₅—C(═O)NH—), alkoxy,alkylamino, a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to the aryl or heteroaryl ring.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to 0, and substituted or unsubstituted loweralkyl.

In certain embodiments, R₃ includes a substituent, such as a substitutedor unsubstituted alkyl or a halogen, at a position para either to X orto the ring including A.

In certain embodiments, the subject compound is selected from thecompounds depicted in FIG. 32.

As described in further detail below, it is contemplated that thesubject methods can be carried out using a variety of different smallmolecules which can be readily identified, for example, by such drugscreening assays as described herein. For example, compounds useful inthe subject methods include compounds may be represented by generalformula (III):

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)—, or a direct bond, preferably —N(R₇)—, —O—, —S—, or a directbond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring;

Q is absent, or represents CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl;

Ar represents a substituted or unsubstituted aryl or heteroaryl ring,such as a substituted or unsubstituted phenyl ring;

R₁ represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl,aryl, heteroaryl, heterocyclyl, or cycloalkyl, including polycyclicgroups;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X—Y—Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes an aryl or heteroaryl ring,optionally substituted with from 1-5 substituents, such as nitro,halogen, cyano, lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy,alkylamino, a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to the aryl or heteroaryl ring.

In certain embodiments, X and the ring comprising A are disposed on Arin a meta (i.e., 1,3) relationship.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, the subject compound is selected from thecompounds depicted in FIG. 32.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (IV):

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —ON(R₇)—, or adirect bond, preferably —N(R₇), —O—, —S—, or a direct bond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring.

Q is absent, or represents CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl;

R₁ represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl,aryl, heteroaryl, heterocyclyl, or cycloalkyl, including polycyclicgroups;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, S-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₃ represents from 0-4 substituents on the ring to which it is attached,such as halogen, hydroxyl, alkoxy, amino, alkylamino, cyano, nitro,substituted or unsubstituted lower alkyl, and acyl, preferably halogen,lower alkoxy, substituted or unsubstituted lower alkyl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a Chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X—Y—Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes an aryl or heteroaryl ring,optionally substituted with from 1-5 substituents, such as nitro,halogen, cyano, lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy,alkylamino, a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to the aryl or heteroaryl ring.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, R₃ includes a substituent, such as a substitutedor unsubstituted alkyl or a halogen, at a position para either to X orto the ring including A.

In certain embodiments, the subject compound is selected from thecompounds depicted in FIG. 32.

In certain embodiments, the subject antagonists can be chosen on thebasis of their selectively for the hedgehog pathway. This selectivitycan be for the hedgehog pathway versus other pathways, or forselectivity between particular hedgehog pathways, e.g., ptc-1, ptc-2,etc.

In certain preferred embodiments, the subject inhibitors inhibit ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function mediated signal transduction with an ED₅₀ of 1 mM orless, more preferably of 1 μM or less, and even more preferably of 1 nMor less. Similarly, in certain preferred embodiments, the subjectinhibitors inhibit activity of the hedgehog pathway with a K_(i) lessthan 10 nM, preferably less than 1 nM even more preferably less than 0.1nM.

In particular embodiments, the small molecule is chosen for use becauseit is more selective for one patched isoform over the next, e.g.,10-fold, and more preferably at least 100- or even 1000-fold moreselective for one patched pathway (ptc-1, ptc-2) over another.

In certain embodiments, a compound which is an antagonist of thehedgehog pathway is chosen to selectively antagonize hedgehog activityover protein kinases other than PKA, such as PKC, e.g., the compoundmodulates the activity of the hedgehog pathway at least an order ofmagnitude more strongly than it modulates the activity of anotherprotein kinase, preferably at least two orders of magnitude morestrongly, even more preferably at least three orders of magnitude morestrongly. Thus, for example, a preferred inhibitor of the hedgehogpathway may inhibit hedgehog activity with a K_(i) at least an order ofmagnitude lower than its K_(i) for inhibition of PKC, preferably atleast two orders of magnitude lower, even more preferably at least threeorders of magnitude lower. In certain embodiments, the K_(i) for PKAinhibition is less than 10 nM, preferably less than 1 nM, even morepreferably less than 0.1 nM.

IV. Exemplary Applications of Method and Compositions

Another aspect of the present invention relates to a method ofmodulating a differentiated state, survival, and/or proliferation of acell having a ptc loss-of-function, hedgehog gain-of-function, orsmoothened gain-of-function, by contacting the cells with a hedgehogantagonist according to the subject method and as the circumstances maywarrant.

For instance, it is contemplated by the invention that, in light of thefindings of an apparently broad involvement of hedgehog, ptc, andsmoothened in the formation of ordered spatial arrangements ofdifferentiated tissues in vertebrates, the subject method could be usedas part of a process for generating and/or maintaining an array ofdifferent vertebrate tissue both in vitro and in vivo. The hedgehogantagonist, whether inductive or anti-inductive with respectproliferation or differentiation of a given tissue, can be, asappropriate, any of the preparations described above.

For example, the present method is applicable to cell culture techniqueswherein, whether for genetic or biochemical reasons, the cells have aptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function phenotype. In vitro neuronal culture systems haveproved to be fundamental and indispensable tools for the study of neuraldevelopment, as well as the identification of neurotrophic factors suchas nerve growth factor (NCF), ciliary trophic factors (CNTF), and brainderived neurotrophic factor (BDNF). One use of the present method may bein cultures of neuronal stem cells, such as in the use of such culturesfor the generation of new neurons and glia. In such embodiments of thesubject method, the cultured cells can be contacted with a hedgehogantagonist of the present invention in order to alter the rate ofproliferation of neuronal stem cells in the culture and/or alter therate of differentiation, or to maintain the integrity of a culture ofcertain terminally differentiated neuronal cells. In an exemplaryembodiment, the subject method can be used to culture, for example,sensory neurons or, alternatively, motorneurons. Such neuronal culturescan be used as convenient assay systems as well as sources ofimplantable cells for therapeutic treatments.

According to the present invention, large numbers of non-tumorigenicneural progenitor cells can be perpetuated in vitro and their rate ofproliferation and/or differentiation can be affected by contact withhedgehog antagonists of the present invention. Generally, a method isprovided comprising the steps of isolating neural progenitor cells froman animal, perpetuating these cells in vitro or in vivo, preferably inthe presence of growth factors, and regulating the differentiation ofthese cells into particular neural phenotypes, e.g., neurons and glia,by contacting the cells with a hedgehog antagonist.

Progenitor cells are thought to be under a tonic inhibitory influencethat maintains the progenitors in a suppressed state until theirdifferentiation is required. However, recent techniques have beenprovided which permit these cells to be proliferated, and unlike neuronsthat are terminally differentiated and therefore non dividing, they canbe produced in unlimited number and are highly suitable fortransplantation into heterologous and autologous hosts withneurodegenerative diseases.

By “progenitor” it is meant an oligopotent or multipotent stem cell thatis able to divide without limit and, under specific conditions, canproduce daughter cells that terminally differentiate such as intoneurons and glia. These cells can be used for transplantation into aheterologous or autologous host. By heterologous is meant a host otherthan the animal from which the progenitor cells were originally derived.By autologous is meant the identical host from which the cells wereoriginally derived.

Cells can be obtained from embryonic, post-natal, juvenile or adultneural tissue from any animal. By any animal is meant any multicellularanimal that contains nervous tissue. More particularly, is meant anyfish, reptile, bird, amphibian or mammal and the like. The mostpreferable donors are mammals, especially mice and humans.

In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will serve to restorefunction to a degenerated area of the host's brain. These regionsinclude areas of the central nervous system (CNS) including the cerebralcortex, cerebellum, midbrain, brainstem, spinal cord and ventriculartissue, and areas of the peripheral nervous system (PNS) including thecarotid body and the adrenal medulla. More particularly, these areasinclude regions in the basal ganglia, preferably the striatum whichconsists of the caudate and putamen, or various cell groups such as theglobus pallidus, the subthalamic nucleus, the nucleus basalis which isfound to be degenerated in Alzheimer's Disease patients, or thesubstantia nigra pars compacta Which is found to be degenerated inParkinson's Disease patients.

Human heterologous neural progenitor cells may be derived from fetaltissue obtained from elective abortion, or from a post-natal, juvenileor adult organ donor. Autologous neural tissue can be obtained bybiopsy, or from patients undergoing neurosurgery in which neural tissueis removed, in particular during epilepsy surgery, and more particularlyduring temporal lobectomies and hippo campalectomies.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedure, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrumentor by mincing with a scalpel to a allow outgrowth of specific cell typesfrom a tissue. Dissociation of fetal cells can be carried out in tissueculture medium, while a preferable medium for dissociation of juvenileand adult cells is artificial cerebral spinal fluid (aCSF). Regular aCSFcontains 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃,and 10 mM D-glucose. Low Ca²⁺ aCSF contains the same ingredients exceptfor MgCl₂ at a concentration of 3.2 mM and CaCl₂ at a concentration of0.1 mM.

Dissociated cells can be placed into any known culture medium capable ofsupporting cell growth, including MEM, DMEM, RPMI, F-12, and the like,containing supplements which are required for cellular metabolism suchas glutamine and other amino acids, vitamins, minerals and usefulproteins such as transferrin and the like. Medium may also containantibiotics to prevent contamination with yeast, bacteria and fungi suchas penicillin, streptomycin, gentamicin and the like. In some cases, themedium may contain serum derived from bovine, equine, chicken and thelike. A particularly preferable medium for cells is a mixture of DMEMand F-12.

Conditions for culturing should be close to physiological conditions.The pH of the culture media should be close to physiological pH,preferably between pH 6-8, more preferably close to pH 7, even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.-40 DC,more preferably between 32° C.-38 DC, and most preferably between 35°C.-37° C.

Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al, (1992) Science 255:1070-4709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating for splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growthfactor which allows for the proliferation of progenitor cells and seededin any receptacle capable of sustaining cells, though as set out above,preferably in culture flasks or roller bottles. Cells typicallyproliferate within 3-4 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask andcontinue to proliferate in suspension forming a hollow sphere ofundifferentiated cells. After approximately 3-10 days in vitro, theproliferating clusters (neurospheres) are fed every 2-7 days, and moreparticularly every 2-4 days by gentle centrifugation and resuspension inmedium containing growth factor.

After 6-7 days in vitro, individual cells in the neurospheres can beseparated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be control in culture by plating (or resuspending) the cellsin the presence of a hedgehog antagonist.

To further illustrate other uses of the subject hedgehog antagonists, itis noted that intracerebral grafting has emerged as an additionalapproach to central nervous system therapies. For example, one approachto repairing damaged brain tissues involves the transplantation of cellsfrom fetal or neonatal animals into the adult brain (Dunnett et al.(1987) J Exp Biot 123:265-289; and Freund et al, (1985) J Neurosci 5;603-616). Fetal neurons from a variety of brain regions can besuccessfully incorporated into the adult brain, and such grafts canalleviate behavioral defects. For example, movement disorder induced bylesions of dopaminergic projections to the basal ganglia can beprevented by grafts of embryonic dopaminergic neurons. Complex cognitivefunctions that are impaired after lesions of the neocortex can also bepartially restored by grafts of embryonic cortical cells. The subjectmethod can be used to regulate the growth state in the culture, or wherefetal tissue is used, especially neuronal stem cells, can be used toregulate the rate of differentiation of the stem cells.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells, andothers of which can generate only one type of cell, such as sensoryneurons, and likely represent committed progenitor cells. The role ofhedgehog antagonists employed in the present method to culture such stemcells can be to regulate differentiation of the uncommitted progenitor,or to regulate further restriction of the developmental fate of acommitted progenitor cell towards becoming a terminally differentiatedneuronal cell. For example, the present method can be used in vitro toregulate the differentiation of neural crest cells into glial cells,schwann cells, chromaffin cells, cholinergic sympathetic orparasympathetic neurons, as well as peptidergic and serotonergicneurons. The hedgehog antagonists can be used alone, or can be used incombination with other neurotrophic factors that act to moreparticularly enhance a particular differentiation fate of the neuronalprogenitor cell.

In addition to the implantation of cells cultured in the presence of thesubject hedgehog antagonists, yet another aspect of the presentinvention concerns the therapeutic application of a hedgehog antagonistto regulate the growth state of neurons and other neuronal cells in boththe central nervous system and the peripheral nervous system. Theability of ptc, hedgehog, and smoothened to regulate neuronaldifferentiation during development of the nervous system and alsopresumably in the adult state indicates that, in certain instances, thesubject hedgehog antagonists can be expected to facilitate control ofadult neurons with regard to maintenance, functional performance, andaging of normal cells; repair and regeneration processes in chemicallyor mechanically lesioned cells; and treatment of degeneration in certainpathological conditions. In light of this understanding, the presentinvention specifically contemplates applications of the subject methodto the treatment protocol of (prevention and/or reduction of theseverity of) neurological conditions deriving from: (i) acute, subacute,or chronic injury to the nervous system, including traumatic injury,chemical injury, vascular injury and deficits (such as the ischemiaresulting from stroke), together with infectious/inflammatory andtumor-induced injury; (ii) aging of the nervous system includingAlzheimer's disease; (iii) chronic neurodegenerative diseases of thenervous system, including Parkinson's disease, Huntington's chorea,amylotrophic lateral sclerosis and the like, as well as spinocerebellardegenerations; and (iv) chronic immunological diseases of the nervoussystem or affecting the nervous system, including multiple sclerosis.

As appropriate, the subject method can also be used in generating nerveprostheses for the repair of central and peripheral nerve damage. Inparticular, where a crushed or severed axon is intubulated by use of aprosthetic device, hedgehog antagonists can be added to the prostheticdevice to regulate the rate of growth and regeneration of the dendridicprocesses. Exemplary nerve guidance channels are described in U.S. Pat.Nos. 5,092,871 and 4,955,892.

In another embodiment, the subject method can be used in the treatmentof neoplastic or hyperplastic transformations such as may occur in thecentral nervous system. For instance, the hedgehog antagonists can beutilized to cause such transformed cells to become either post-mitoticor apoptotic. The present method may, therefore, be used as part of atreatment for, e.g., malignant gliomas, meningiomas, medulloblastomas,neuroectodermal tumors, and ependymomas.

In a preferred embodiment, the subject method can be used as part of atreatment regimen for malignant medulloblastoma and other primary CNSmalignant neuroectodermal tumors.

In certain embodiments, the subject method is used as part of treatmentprogram for medulloblastoma. Medulloblastoma, a primary brain tumor, isthe most common brain tumor in children. A medulloblastoma is aprimitive neuroectodermal tumor arising in the posterior fossa. Theyaccount for approximately 25% of all pediatric brain tumors (Miller).Histologically, they are small round cell tumors commonly arranged intrue rosettes, but may display some differentiation to astrocytes,ependymal cells or neurons (Rorke; Kleihues), PNET's may arise in otherareas of the brain including the pineal gland (pineoblastoma) andcerebrum. Those arising in the supratentorial region generally fareworse than their PF counterparts.

Medulloblastoma/PNET's are known to recur anywhere in the CNS afterresection, and can even metastasize to bone. Pretreatment evaluationshould therefore include an examination of the spinal cord to excludethe possibility of “dropped metastases”. Gadolinium-enhanced MRI haslargely replaced myelography for this purpose, and CSF cytology isobtained postoperatively as a routine procedure.

In other embodiments, the subject method is used as part of treatmentprogram for ependymomas. Ependymomas account for approximately 10% ofthe pediatric brain tumors in children. Grossly, they are tumors thatarise from the ependymal lining of the ventricles and microscopicallyform rosettes, canals, and perivascular rosettes. In the CHOP series of51 children reported with ependymomas, ¾ were histologically benign.Approximately ⅔ arose from the region of the 4th ventricle. One thirdpresented in the supratentorial region. Age at presentation peaksbetween birth and 4 years, as demonstrated by SEER data as well as datafrom CHOP. The median age is about 5 years. Because so many childrenwith this disease are babies, they often require multimodal therapy.

Yet another aspect of the present invention concerns the observation inthe art that ptc, hedgehog, and/or smoothened are involved inmorphogenic signals involved in other vertebrate organogenic pathways inaddition to neuronal differentiation as described above, having apparentroles in other endodermal patterning, as well as both mesodermal andendodermal differentiation processes. Thus, it is contemplated by theinvention that compositions comprising hedgehog antagonists can also beutilized for both cell culture and therapeutic methods involvinggeneration and maintenance of non-neuronal tissue.

In one embodiment, the present invention makes use of the discovery thatptc, hedgehog, and smoothened are apparently involved in controlling thedevelopment of stem cells responsible for formation of the digestivetract, liver, lungs, and other organs which derive from the primitivegut. Shh serves as an inductive signal from the endoderm to themesoderm, which is critical to gut morphogenesis. Therefore, forexample, hedgehog antagonists of the instant method can be employed forregulating the development and maintenance of an artificial liver thatcan have multiple metabolic functions of a normal liver. In an exemplaryembodiment, the subject method can be used to regulate the proliferationand differentiation of digestive tube stem cells to form hepatocytecultures which can be used to populate extracellular matrices, or whichcan be encapsulated in biocompatible polymers, to form both implantableand extracorporeal artificial livers.

In another embodiment, therapeutic compositions of hedgehog antagonistscan be utilized in conjunction with transplantation of such artificiallivers, as well as embryonic liver structures, to regulate uptake ofintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

In yet another embodiment, the subject method can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisinghedgehog antagonists can be utilized in liver repair subsequent to apartial hepatectomy.

The generation of the pancreas and small intestine from the embryonicgut depends on intercellular signalling between the endodermal andmesodermal cells of the gut. In particular, the differentiation ofintestinal mesoderm into smooth muscle has been suggested to depend onsignals from adjacent endodermal cells. One candidate mediator ofendodermally derived signals in the embryonic hindgut is Sonic hedgehog.See, for example, Apelqvist et al, (1997) Curr Biol 7:801-4. The Shhgene is expressed throughout the embryonic gut endoderm with theexception of the pancreatic bud endoderm, which instead expresses highlevels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor1/pancreatic and duodenal homeobox 1), an essential regulator of earlypancreatic development. Apelqvist et al., supra, have examined whetherthe differential expression of Shh in the embryonic gut tube controlsthe differentiation of the surrounding mesoderm into specialisedmesoderm derivatives of the small intestine and pancreas. To test this,they used the promoter of the Ipf1/Pdx1 gene to selectively express Shhin the developing pancreatic epithelium. In Ipf1/Pdx1-Shh transgenicmice, the pancreatic mesoderm developed into smooth muscle andinterstitial cells of Cajal, characteristic of the intestine, ratherthan into pancreatic mesenchyme and spleen. Also, pancreatic explantsexposed to Shh underwent a similar program of intestinaldifferentiation. These results provide evidence that the differentialexpression of endodermally derived Shh controls the fate of adjacentmesoderm at different regions of the gut tube.

In the context of the present invention, it is contemplated thereforethat the subject hedgehog antagonists can be used to control or regulatethe proliferation and/or differentiation of pancreatic tissue both invivo and in vitro.

There are a wide variety of pathological cell proliferative anddifferentiative conditions for which the inhibitors of the presentinvention may provide therapeutic benefits, with the general strategybeing, for example, the correction of aberrant insulin expression, ormodulation of differentiation. More generally, however, the presentinvention relates to a method of inducing and/or maintaining adifferentiated state, enhancing survival and/or affecting proliferationof pancreatic cells, by contacting the cells with the subjectinhibitors. For instance, it is contemplated by the invention that, inlight of the apparent involvement of ptc, hedgehog, and smoothened inthe formation of ordered spatial arrangements of pancreatic tissues, thesubject method could be used as part of a technique to generate and/ormaintain such tissue both in vitro and in vivo. For instance, modulationof the function of hedgehog can be employed in both cell culture andtherapeutic methods involving generation and maintenance β-cells andpossibly also for non-pancreatic tissue, such as in controlling thedevelopment and maintenance of tissue from the digestive tract, spleen,lungs, urogenital organs (e.g., bladder), and other organs which derivefrom the primitive gut.

In an exemplary embodiment, the present method can be used in thetreatment of hyperplastic and neoplastic disorders effecting pancreatictissue, particularly those characterized by aberrant proliferation ofpancreatic cells. For instance, pancreatic cancers are marked byabnormal proliferation of pancreatic cells that can result inalterations of insulin secretory capacity of the pancreas. For instance,certain pancreatic hyperplasias, such as pancreatic carcinomas, canresult in hypoinsulinemia due to dysfunction of β-cells or decreasedislet cell mass. To the extent that aberrant ptc, hedgehog, andsmoothened signaling may be indicated in disease progression, thesubject inhibitors, can be used to enhance regeneration of the tissueafter anti-tumor therapy.

Moreover, manipulation of hedgehog signaling properties at differentpoints may be useful as part of a strategy for reshaping/repairingpancreatic tissue both in vivo and in vitro. In one embodiment, thepresent invention makes use of the apparent involvement of ptc,hedgehog, and smoothened in regulating the development of pancreatictissue, in general, the subject method can be employed therapeuticallyto regulate the pancreas after physical, chemical or pathologicalinsult. In yet another embodiment, the subject method can be applied tocell culture techniques, and in particular, may be employed to enhancethe initial generation of prosthetic pancreatic tissue devices.Manipulation of proliferation and differentiation of pancreatic tissue,for example, by altering hedgehog activity, can provide a means for morecarefully controlling the characteristics of a cultured tissue. In anexemplary embodiment, the subject method can be used to augmentproduction of prosthetic devices which require β-islet cells, such asmay be used in the encapsulation devices described in, for example, theAebischer et al, U.S. Pat. No. 4,892,538, the Aebischer et al. U.S. Pat.No. 5,106,627, the Lim U.S. Pat. No. 4,391,909, and the Sefton U.S. Pat.No. 4,353,888. Early progenitor cells to the pancreatic islets aremultipotential, and apparently coactivate all the islet-specific genesfrom the time they first appear. As development proceeds, expression ofislet-specific hormones, such as insulin, becomes restricted to thepattern of expression characteristic of mature islet cells. Thephenotype of mature islet cells, however, is not stable in culture, asreappearance of embryonal traits in mature β-cells can be observed. Byutilizing the subject hedgehog antagonists, the differentiation path orproliferative index of the cells can be regulated.

Furthermore, manipulation of the differentiative state of pancreatictissue can be utilized in conjunction with transplantation of artificialpancreas so as to promote implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted tissue. For instance,manipulation of hedgehog function to affect tissue differentiation canbe utilized as a means of maintaining graft viability.

Bellusci et al. (1997) Development 124:53 report that Sonic hedgehogregulates lung mesenchymal cell proliferation in vivo. Accordingly, thepresent method can be used to regulate regeneration of lung tissue,e.g., in the treatment of emphysema and respiratory distress syndrome.

Fujita et al. (1997) Biochem Biophys Res Commun 238:658 reported thatSonic hedgehog is expressed in human lung squamous carcinoma andadenocarcinoma cells. The expression of Sonic hedgehog was also detectedin the human lung squamous carcinoma tissues, but not in the normal lungtissue of the same patient. They also observed that Sonic hedgehogstimulates the incorporation of BrdU into the carcinoma cells andstimulates their cell growth, while anti-Shh-N inhibited their cellgrowth. These results suggest that a ptc, hedgehog, and/or smoothened isinvolved in the cell growth of such transformed lung tissue andtherefore indicates that the subject method can be used as part of atreatment of lung carcinoma and adenocarcinomas, and other proliferativedisorders involving the lung epithelia.

Many other tumors may, based on evidence such as involvement of thehedgehog pathway in these tumors, or detected expression of hedgehog orits receptor in these tissues during development, be affected bytreatment with the subject compounds. Such tumors include, but are by nomeans limited to, tumors related to Gorlin's syndrome (e.g., basal cellcarcinoma, medulloblastoma, meningioma, etc.), tumors evidenced in petknock-out mice (e.g., bernangioma, rhabdomyosarcoma, etc.), tumorsresulting from gli-1 amplification (e.g., glioblastoma, sarcoma, etc.),tumors connected with TRC8, a ptc homolog (e.g., renal carcinoma,thyroid carcinoma, etc.), Ext-1-related tumors (e.g., bone cancer,etc.), Shh-induced tumors (e.g., lung cancer, chondrosarcomas, etc.),and other tumors (e.g., breast cancer, urogenital cancer (e.g., kidney,bladder, ureter, prostate, etc.), adrenal cancer, gastrointestinalcancer (e.g., stomach, intestine, etc.), etc.).

In still another embodiment of the present invention, compositionscomprising hedgehog antagonists can be used in the in vitro generationof skeletal tissue, such as from skeletogenic stem cells, as well as thein vivo treatment of skeletal tissue deficiencies. The present inventionparticularly contemplates the use of hedgehog antagonists to regulatethe rate of chondrogenesis and/or osteogenesis. By “skeletal tissuedeficiency”, it is meant a deficiency in bone or other skeletalconnective tissue at any site where it is desired to restore the bone orconnective tissue, no matter how the deficiency originated, e.g. whetheras a result of surgical intervention, removal of tumor, ulceration,implant, fracture, or other traumatic or degenerative conditions.

For instance, the method of the present invention can be used as part ofa regimen for restoring cartilage function to a connective tissue. Suchmethods are useful in, for example, the repair of defects or lesions incartilage tissue which is the result of degenerative wear such as thatwhich results in arthritis, as well as other mechanical derangementswhich may be caused by trauma to the tissue, such as a displacement oftorn meniscus tissue, meniscectomy, a laxation of a joint by a tornligament, malignment of joints, bone fracture, or by hereditary disease.The present reparative method is also useful for remodeling cartilagematrix, such as in plastic or reconstructive surgery, as well asperiodontal surgery. The present method may also be applied to improvinga previous reparative procedure, for example, following surgical repairof a meniscus, ligament, or cartilage. Furthermore, it may prevent theonset or exacerbation of degenerative disease if applied early enoughafter trauma.

In one embodiment of the present invention, the subject method comprisestreating the afflicted connective tissue with a therapeuticallysufficient amount of a hedgehog antagonist, particularly an antagonistselective for Indian hedgehog signal transduction, to regulate acartilage repair response in the connective tissue by managing the rateof differentiation and/or proliferation of chondrocytes embedded in thetissue. Such connective tissues as articular cartilage, interarticularcartilage (menisci), costal cartilage (connecting the true ribs and thesternum), ligaments, and tendons are particularly amenable to treatmentin reconstructive and/or regenerative therapies using the subjectmethodAs used herein, regenerative therapies include treatment ofdegenerative states which have progressed to the point of whichimpairment of the tissue is obviously manifest, as well as preventivetreatments of tissue where degeneration is in its earliest stages orimminent.

In an illustrative embodiment, the subject method can be used as part ofa therapeutic intervention in the treatment of cartilage of adiarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist,a knuckle of either a finger or toe, or a tempomandibular joint. Thetreatment can be directed to the meniscus of the joint, to the articularcartilage of the joint, or both. To further illustrate, the subjectmethod can be used to treat a degenerative disorder of a knee, such aswhich might be the result of traumatic injury (e.g., a sports injury orexcessive wear) or osteoarthritis. The subject antagonists may beadministered as an injection into the joint with, for instance, anarthroscopic needle. In sonic instances, the injected agent can be inthe form of a hydrogel or other slow release vehicle described above inorder to permit a more extended and regular contact of the agent withthe treated tissue.

The present invention further contemplates the use of the subject methodin the field of cartilage transplantation and prosthetic devicetherapies. However, problems arise, for instance, because thecharacteristics of cartilage and fibrocartilage varies between differenttissue: such as between articular, meniscal cartilage, ligaments, andtendons, between the two ends of the same ligament or tendon, andbetween the superficial and deep parts of the tissue. The zonalarrangement of these tissues may reflect a gradual change in mechanicalproperties, and failure occurs when implanted tissue, which has notdifferentiated under those conditions, lacks the ability toappropriately respond. For instance, when meniscal cartilage is used torepair anterior cruciate ligaments, the tissue undergoes a metaplasia topure fibrous tissue. By regulating the rate of chondrogenesis, thesubject method can be used to particularly address this problem, byhelping to adaptively control the implanted cells in the new environmentand effectively resemble hypertrophic chondrocytes of an earlierdevelopmental stage of the tissue.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. The need for improved treatment has motivated researchaimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 7113:74; Vacanti et al, (1991) Plast Reconsir Surg88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed etal. (1993) J Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scaffolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers that degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own ear or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

In one embodiment of the subject method, the implants are contacted witha hedgehog antagonist during certain stages of the culturing process inorder to manage the rate of differentiation of chondrocytes and theformation of hypertrophic chrondrocytes in the culture.

in another embodiment, the implanted device is treated with a hedgehogantagonist in order to actively remodel the implanted matrix and to makeit more suitable for its intended function. As set out above withrespect to tissue transplants, the artificial transplants suffer fromthe same deficiency of not being derived in a setting which iscomparable to the actual mechanical environment in which the matrix isimplanted. The ability to regulate the chondrocytes in the matrix by thesubject method can allow the implant to acquire characteristics similarto the tissue for which it is intended to replace.

In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis.

In still further embodiments, the subject method can be employed as partof a regimen for the generation of bone (osteogenesis) at a site in theanimal where such skeletal tissue is deficient. Indian hedgehog isparticularly associated with the hypertrophic chondrocytes that areultimately replaced by osteoblasts. For instance, administration of ahedgehog antagonist of the present invention can be employed as part ofa method for regulating the rate of bone loss in a subject. For example,preparations comprising hedgehog antagonists can be employed, forexample, to control endochondral ossification in the formation of a“model” for ossification.

In yet another embodiment of the present invention, a hedgehogantagonist can be used to regulate spermatogenesis. The hedgehogproteins, particularly Dhh, have been shown to be involved in thedifferentiation and/or proliferation and maintenance of testicular germcells. Dhh expression is initiated in Sertoli cell precursors shortlyafter the activation of Sry (testicular determining gene) and persistsin the testis into the adult. Males are viable but infertile, owing to acomplete absence of mature sperm. Examination of the developing testisin different genetic backgrounds suggests that Dhh regulates both earlyand late stages of spermatogenesis. Bitgood et al. (1996) Curr Biol6:298. In a preferred embodiment, the hedgehog antagonist can be used asa contraceptive. In similar fashion, hedgehog antagonists of the subjectmethod are potentially useful for modulating normal ovarian function.

The subject method also has wide applicability to the treatment orprophylaxis of disorders afflicting epithelial tissue, as well as incosmetic uses. In general, the method can be characterized as includinga step of administering to an animal an amount of a hedgehog antagonisteffective to alter the growth state of a treated epithelial tissue. Themode of administration and dosage regimens will vary depending on theepithelial tissue(s) to be treated. For example, topical formulationswill be preferred where the treated tissue is epidermal tissue, such asdermal or mucosal tissues.

A method that “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method regulates the proliferation and/or growth of,ruler alia, keratinocytes, or that the wound heals with less scarring,less wound contraction, less collagen deposition and more superficialsurface area. In certain instances, “promotion of wound healing” canalso mean that certain methods of wound healing have improved successrates, (e.g., the take rates of skin grafts) when used together with themethod of the present invention.

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of scar tissue.

The present treatment can also be effective as part of a therapeuticregimen for treating oral and paraoral ulcers, e.g. resulting fromradiation and/or chemotherapy. Such ulcers commonly develop within daysafter chemotherapy or radiation therapy. These ulcers usually begin assmall, painful irregularly shaped lesions usually covered by a delicategray necrotic membrane and surrounded by inflammatory tissue. In manyinstances, lack of treatment results in proliferation of tissue aroundthe periphery of the lesion on an inflammatory basis. For instance, theepithelium bordering the ulcer usually demonstrates proliferativeactivity, resulting in loss of continuity of surface epithelium. Theselesions, because of their size and loss of epithelial integrity, disposethe body to potential secondary infection. Routine ingestion of food andwater becomes a very painful event and, if the ulcers proliferatethroughout the alimentary canal, diarrhea usually is evident with allits complicating actors. According to the present invention, a treatmentfor such ulcers which includes application of a hedgehog antagonist canreduce the abnormal proliferation and differentiation of the affectedepithelium, helping to reduce the severity of subsequent inflammatoryevents.

The subject method and compositions can also be used to treat woundsresulting from dermatological diseases, such as lesions resulting fromautoimmune disorders such as psoriasis. Atopic dermititis refers to skintrauma resulting from allergies associated with an immune responsecaused by allergens such as pollens, foods, dander, insect venoms andplant toxins.

In other embodiments, antiproliferative preparations of hedgehogantagonists can be used to inhibit lens epithelial cell proliferation toprevent post-operative complications of extracapsular cataractextraction. Cataract is an intractable eye disease and various studieson a treatment of cataract have been made. But at present, the treatmentof cataract is attained by surgical operations. Cataract surgery hasbeen applied for a long time and various operative methods have beenexamined. Extracapsular lens extraction has become the method of choicefor removing cataracts. The major medical advantages of this techniqueover intracapsular extraction are lower incidence of aphakic cystoidmacular edema and retinal detachment. Extracapsular extraction is alsorequired for implantation of posterior chamber type intraocular lensesthat are now considered to be the lenses of choice in most cases.

However, a disadvantage of extracapsular cataract extraction is the highincidence of posterior lens capsule opacification, often calledafter-cataract, which can occur in up to 50% of cases within three yearsafter surgery. After-cataract is caused by proliferation of equatorialand anterior capsule lens epithelial cells that remain afterextracapsular lens extraction. These cells proliferate to causeSommerling rings, and along with fibroblasts that also deposit and occuron the posterior capsule, cause opacification of the posterior capsule,which interferes with vision. Prevention of after-cataract would bepreferable to treatment. To inhibit secondary cataract formation, thesubject method provides a means for inhibiting proliferation of theremaining lens epithelial cells. For example, such cells can be inducedto remain quiescent by instilling a solution containing an hedgehogantagonist preparation into the anterior chamber of the eye after lensremoval. Furthermore, the solution can be osmotically balanced toprovide minimal effective dosage when instilled into the anteriorchamber of the eye, thereby inhibiting stibcapsular epithelial growthwith some specificity.

The subject method can also be used in the treatment of corneopathiesmarked by corneal epithelial cell proliferation, as for example inocular epithelial disorders such as epithelial dowrigrowth or squamouscell carcinomas of the ocular surface.

Levine et al. (1997) J Neurosci 17:6277 show that hedgehog proteins canregulate mitogenesis and photoreceptor differentiation in the vertebrateretina, and Ihh is a candidate factor from the pigmented epithelium topromote retinal progenitor proliferation and photoreceptordifferentiation. Likewise, Jensen et al. (1997) Development 124:363demonstrated that treatment of cultures of perinatal mouse retinal cellswith the amino-terminal fragment of Sonic hedgehog protein results in anincrease in the proportion of cells that incorporate bromodeoxuridine,in total cell numbers, and in rod photoreceptors, amacrine cells andMuller glial cells, suggesting that Sonic hedgehog promotes theproliferation of retinal precursor cells. Thus, the subject method canbe used in the treatment of proliferative diseases of retinal cells andregulate photoreceptor differentiation.

Yet another aspect of the present invention relates to the use of thesubject method to control hair growth. Flair is basically composed ofkeratin, a tough and insoluble protein; its chief strength lies in itsdisulphide bond of cystine. Each individual hair comprises a cylindricalshaft and a root, and is contained in a follicle, a flask-likedepression in the skin. The bottom of the follicle contains afinger-like projection termed the papilla, which consists of connectivetissue from which hair grows, and through which blood vessels supply thecells with nourishment. The shaft is the part that extends outwards fromthe skin surface, whilst the root has been described as the buried partof the hair. The base of the root expands into the hair bulb, whichrests upon the papilla. Cells from which the hair is produced grow inthe bulb of the follicle; they are extruded in the form of fibers as thecells proliferate in the follicle. Hair “growth” refers to the formationand elongation of the hair fiber by the dividing cells.

As is well known in the art, the common hair cycle is divided into threestages: anagen, catagen and telogen. During the active phase (anagen),the epidermal stem cells of the dermal papilla divide rapidly. Daughtercells move upward and differentiate to form the concentric layers of thehair itself. The transitional stage, catagen, is marked by the cessationof mitosis of the stem cells in the follicle. The resting stage is knownas telogen, where the hair is retained within the scalp for severalweeks before an emerging new hair developing below it dislodges thetelogen-phase shaft from its follicle. From this model it has becomeclear that the larger the pool of dividing stem cells that differentiateinto hair cells, the more hair growth occurs. Accordingly, methods forincreasing or reducing hair growth can be carried out by potentiating orinhibiting, respectively, the proliferation of these stem cells.

In certain embodiments, the subject method can be employed as a way ofreducing the growth of human hair as opposed to its conventional removalby cutting, shaving, or depilation. For instance, the present method canbe used in the treatment of trichosis characterized by abnormally rapidor dense growth of hair, e.g. hypertrichosis. In an exemplaryembodiment, hedgehog antagonists can be used to manage hirsutism, adisorder marked by abnormal hairiness. The subject method can alsoprovide a process for extending the duration of depilation.

Moreover, because a hedgehog antagonist will often be cytostatic toepithelial cells, rather than cytotoxic, such agents can be used toprotect hair follicle cells from cytotoxic agents that requireprogression into S-phase of the cell-cycle for efficacy, e.g.radiation-induced death. Treatment by the subject method can provideprotection by causing the hair follicle cells to become quiescent, e.g.,by inhibiting the cells from entering S phase, and thereby preventingthe follicle cells from undergoing mitotic catastrophe or programmedcell death. For instance, hedgehog antagonists can be used for patientsundergoing chemo- or radiation-therapies that ordinarily result in hairloss. By inhibiting cell-cycle progression during such therapies, thesubject treatment can protect hair follicle cells from death that mightotherwise result from activation of cell death programs. After thetherapy has concluded, the instant method can also be removed withconcommitant relief of the inhibition of follicle cell proliferation.

The subject method can also be used in the treatment of folliculitis,such as folliculitis decalvans, folliculitis ulerythematosa reticulataor keloid folliculitis. For example, a cosmetic preparation of anhedgehog antagonist can be applied topically in the treatment ofpseudofolliculitis, a chronic disorder occurring most often in thesubmandibular region of the neck and associated with shaving, thecharacteristic lesions of which are erythematous papules and pustulescontaining buried hairs.

In another aspect of the invention, the subject method can be used toinduce differentiation and/or inhibit proliferation of epitheliallyderived tissue. Such forms of these molecules can provide a basis fordifferentiation therapy for the treatment of hyperplastic and/orneoplastic conditions involving epithelial tissue. For example, suchpreparations can be used for the treatment of cutaneous diseases inwhich there is abnormal proliferation or growth of cells of the skin.

For instance, the pharmaceutical preparations of the invention areintended for the treatment of hyperplastic epidermal conditions, such askeratosis, as well as for the treatment of neoplastic epidermalconditions such as those characterized by a high proliferation rate forvarious skin cancers, as for example basal cell carcinoma or squamouscell carcinoma. The subject method can also be used in the treatment ofautoimmune diseases affecting the skin, in particular, of dermatologicaldiseases involving morbid proliferation and/or keratinization of theepidermis, as for example, caused by psoriasis or atopic dermatosis.

Many common diseases of the skin, such as psoriasis, squamous cellcarcinoma, keratoacanthoma and actinic keratosis are characterized bylocalized abnormal proliferation and growth. For example, in psoriasis,which is characterized by scaly, red, elevated plaques on the skin, thekeratinocytes are known to proliferate much more rapidly than normal andto differentiate less completely.

In one embodiment, the preparations of the present invention aresuitable for the treatment of dermatological ailments linked tokeratinization disorders causing abnormal proliferation of skin cells,which disorders may be marked by either inflammatory or non-inflammatorycomponents. To illustrate, therapeutic preparations of a hedgehogantagonist, e.g., which promotes quiescense or differentiation can beused to treat varying forms of psoriasis, be they cutaneous, mucosal orungual. Psoriasis, as described above, is typically characterized byepidermal keratinocytes that display marked proliferative activation anddifferentiation along a “regenerative” pathway. Treatment with anantiproliferative embodiment of the subject method can be used toreverse the pathological epidermal activiation and can provide a basisfor sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatmentwith the subject method. Actinic keratoses, for example, are superficialinflammatory premalignant tumors arising on sun-exposed and irradiatedskin. The lesions are erythematous to brown with variable scaling.Current therapies include excisional and cryosurgery. These treatmentsare painful, however, and often produce cosmetically unacceptablescarring. Accordingly, treatment of keratosis, such as actinickeratosis, can include application, preferably topical, of a hedgehogantagonist composition in amounts sufficient to inhibithyperproliferation of epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treated bythe subject method. Acne vulgaris, for instance, is a multifactorialdisease most commonly occurring in teenagers and young adults, and ischaracterized by the appearance of inflammatory and noninflammatorylesions on the face and upper trunk. The basic defect which gives riseto acne vulgaris is hypercornification of the duct of a hyperactivesebaceous gland. Hypercornification blocks the normal mobility of skinand follicle microorganisms, and in so doing, stimulates the release oflipases by Propinohacteriwn acmes and Staphylococcus epidermidisbacteria and Pitrosporum ovale, a yeast. Treatment with anantiproliferative hedgehog antagonist, particularly topicalpreparations, may be useful for preventing the transitional features ofthe ducts, e.g. hypercornification, which lead to lesion formation. Thesubject treatment may further include, for example, antibiotics,retinoids and antiandrogens.

The present invention also provides a method for treating various formsof dermatitis. Dermatitis is a descriptive term referring to poorlydemarcated lesions that are either pruritic, erythematous, scaley,blistered, weeping, fissured or crusted. These lesions arise from any ofa wide variety of causes. The most common types of dermatitis areatopic, contact and diaper dermatitis. For instance, seborrheicdermatitis is a chronic, usually pruritic, dermatitis with erythema,dry, moist, or greasy scaling, and yellow crusted patches on variousareas, especially the scalp, with exfoliation of an excessive amount ofdry scales. The subject method can also be used in the treatment ofstasis dermatitis, an often chronic, usually eczematous dermatitis.Actinic dermatitis is dermatitis that due to exposure to actinicradiation such as that from the sun, ultraviolet waves or x- orgamma-radiation. According to the present invention, the subject methodcan be used in the treatment and/or prevention of certain symptoms ofdermatitis caused by unwanted proliferation of epithelial cells. Suchtherapies for these various forms of dermatitis can also include topicaland systemic corticosteroids, antipuritics, and antibiotics.

For example, it is contemplated that the subject method could be used toinhibit angiogenesis. Hedgehog is known to stimulate angiogenesis.Matrigel plugs impregnated with hedgehog protein and inserted into miceevince substantial neovascularization, whereas Matrigel plugs notcarrying hedgehog show comparatively little vascularization. Hedgehogprotein is also capable of increasing vascularization of the normallyavascular mouse cornea. The ptc-1 gene is expressed in normal vasculartissues, including the endothelial cells of the aorta, vascular smoothmuscle cells, adventitial fibroblasts of the aorta, the coronaryvasculature and cardiomyocytes of the atria and ventricles. Thesetissues are also sensitive to hedgehog protein. Treatment with exogenoushedgehog causes upregulation of ptc-1 expression. In addition, hedgehogproteins stimulate proliferation of vascular smooth muscle cells invivo. Hedgehog proteins also cause fibroblasts to increase expression ofangiogenic growth factors such as VEGF, bFGF, Ang-1 and Ang-2. Lastly,hedgehog proteins are known to stimulate recovery from ischemic injuryand stimulate formation of collateral vessels.

Given that hedgehog promotes angiogenesis, hedgehog antagonists areexpected to act as angiogenesis inhibitors, particularly in situationswhere some level of hedgehog signaling is necessary for angiogenesis.

Angiogenesis is fundamental to many disorders. Persistent, unregulatedangiogenesis occurs in a range of disease states, tumor metastases andabnormal growths by endothelial cells. The vasculature created as aresult of angiogenic processes supports the pathological damage seen inthese conditions. The diverse pathological states created due tounregulated angiogenesis have been grouped together as angiogenicdependent or angiogenic associated diseases. Therapies directed atcontrol of the angiogenic processes could lead to the abrogation ormitigation of these diseases.

Diseases caused by, supported by or associated with angiogenesis includeocular neovascular disease, age-related macular degeneration, diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,neovascular glaucoma, retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,Sjogren's, acne rosacea, phylectemil osis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalUlcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi sarcoma, Mooren ulcer, Terrien's marginaldegeneration, marginal keratolysis, rheumatoid arthritis, systemiclupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, StevensJohnson disease, periphigoid radial keratotomy, corneal graph rejection,rheumatoid arthritis, osteoarthritis chronic inflammation (eg,ulcerative colitis or Crohn's disease), hemangioma, Osler-Weber-Rendudisease, and hereditary hemorrhagic telangiectasia.

In addition, angiogenesis plays a critical role in cancer. A tumorcannot expand without a blood supply to provide nutrients and removecellular wastes. Tumors in which angiogenesis is important include solidtumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma,neuroblastoma, and osteosarcoma, and benign tumors such as acousticneuroma, neurofibroma, trachoma and pyogenic granulomas. Angiogenicfactors have been found associated with several solid tumors. Preventionof angiogenesis could halt the growth of these tumors and the resultantdamage to the animal due to the presence of the tumor. Angiogenesis isalso associated with blood-born tumors such as leukemias, any of variousacute or chronic neoplastic diseases of the bone marrow in whichunrestrained proliferation of white blood cells occurs, usuallyaccompanied by anemia, impaired blood clotting, and enlargement of thelymph nodes, liver, and spleen. It is believed that angiogenesis plays arole in the abnormalities in the bone marrow that give rise toleukemia-like tumors.

In addition to tumor growth, angiogenesis is important in metastasis.Initially, angiogenesis is important is in the vascularization of thetumor which allows cancerous cells to enter the blood stream and tocirculate throughout the body. After the tumor cells have left theprimary site, and have settled into the secondary, metastasis site,angiogenesis must occur before the new tumor can grow and expand.Therefore, prevention of angiogenesis could lead to the prevention ofmetastasis of tumors and possibly contain the neoplastic growth at theprimary site.

Angiogenesis is also involved in normal physiological processes such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation or to prevent implantation by the blastula.

It is anticipated that the invention will be useful for the treatmentand/or prevention of respiratory distress syndrome or other disordersresulting from inappropriate lung surface tension. Respiratory distresssyndrome results from insufficient surfactant in the alveolae of thelungs. The lungs of vertebrates contain surfactant, a complex mixture oflipids and protein that causes surface tension to rise during lunginflation and decrease during lung deflation. During lung deflation,surfactant decreases such that there are no surface forces that wouldotherwise promote alveolar collapse. Aerated alveoli that have notcollapsed during expiration permit continuous oxygen and carbon dioxidetransport between blood and alveolar gas and require much less force toinflate during the subsequent inspiration. During inflation, lungsurfactant increases surface tension as the alveolar surface areaincreases. A rising surface tension in expanding alveoli opposesover-inflation in those airspaces and tends to divert inspired air toless well-aerated alveoli, thereby facilitating even lung aeration.

Respiratory distress syndrome is particularly prevalent among prematureinfants. Lung surfactant is normally synthesized at a very low rateuntil the last six weeks of fetal life. Human infants born more than sixweeks before the normal term of a pregnancy have a high risk of beingborn with inadequate amounts of lung surfactant and inadequate rates ofsurfactant synthesis. The more prematurely an infant is born, the moresevere the surfactant deficiency is likely to be. Severe surfactantdeficiency can lead to respiratory failure within a few minutes or hoursof birth. The surfactant deficiency produces progressive collapse ofalveoli (atelectasis) because of the decreasing ability of the lung toexpand despite maximum inspiratory effort. As a result, inadequateamounts of oxygen reach the infant's blood. RDS can occur in adults aswell, typically as a consequence of failure in surfactant biosynthesis.

Lung tissue of premature infants shows high activity of the hedgehogsignaling pathway. Inhibition of this pathway using hedgehog antagonistsincreases the formation of lamellar bodies and increases the expressionof genes involved in surfactant biosynthesis. Lamellar bodies aresubcellular structures associated with surfactant biosynthesis. Forthese reasons, treatment of premature infants with a hedgehog antagonistshould stimulate surfactant biosynthesis and ameliorate RDS. In caseswhere adult RDS is associated with hedgehog pathway activation,treatment with hedgehog antagonists should also be effective.

It is further contemplated that the use of hedgehog antagonists may bespecifically targeted to disorders where the affected tissue and/orcells evince high hedgehog pathway activation. Expression of gli genesis activated by the hedgehog signaling pathway, including gli-1, gli-2and gli-3. gli-I expression is most consistently correlated withhedgehog signaling activity across a wide range of tissues anddisorders, while gli-3 is somewhat less so. The gil genes encodetranscription factors that activate expression of many genes needed toelicit the full effects of hedgehog signaling. However, the Gli-3transcription factor can also act as a repressor of hedgehog effectorgenes, and therefore, expression of gli-3 can cause a decreased effectof the hedgehog signaling pathway. Whether Gli-3 acts as atranscriptional activator or repressor depends on post-translationalevents, and therefore it is expected that methods for detecting theactivating form (versus the repressing form) of Gli-3 protein would alsobe a reliable measure of hedgehog pathway activation. gli-2 geneexpression is expected to provide a reliable marker for hedgehog pathwayactivation. The gli-1 gene is strongly expressed in a wide array ofcancers, hyperplasias and immature lungs, and serves as a marker for therelative activation of the hedgehog pathway. In addition, tissues, suchas immature lung, that have high gli gene expression are stronglyaffected by hedgehog inhibitors. Accordingly, it is contemplated thatthe detection of gli gene expression may be used as a powerfulpredictive tool to identify tissues and disorders that will particularlybenefit from treatment with a hedgehog antagonist.

In preferred embodiments, gli-1 expression levels are detected, eitherby direct detection of the transcript or by detection of protein levelsor activity. Transcripts may be detected using any of a wide range oftechniques that depend primarily on hybridization of probes to the gli-1transcripts or to cDNAs synthesized therefrom. Well known techniquesinclude Northern blotting, reverse-transcriptase PCR and microarrayanalysis of transcript levels. Methods for detecting Gli protein levelsinclude Western blotting, immunoprecipitation, two-dimensionalpolyacrylamide gel electrophoresis (2D SDS-PAGE) (preferably comparedagainst a standard wherein the position of the Gli proteins has beendetermined), and mass spectroscopy. Mass spectroscopy may be coupledwith a series of purification steps to allow high-throughputidentification of many different protein levels in a particular sample.Mass spectroscopy and 2D SDS-PAGE can also be used to identifypost-transcriptional modifications to proteins including proteolyticevents, ubiquitination, phosphorylation, lipid modification etc. Gliactivity may also be assessed by analyzing binding to substrate DNA orin vitro transcriptional activation of target promoters. Gel shiftassays, DNA footprinting assays and DNA-protein crosslinking assays areall methods that may be used to assess the presence of a protein capableof binding to Gli binding sites on DNA.

In preferred embodiments, gli transcript levels are measured anddiseased or disordered tissues showing abnormally high gil levels aretreated with a hedgehog antagonist. Premature lung tissue, lung cancers(e.g., adenocarcinomas, broncho-alveolar adenocarcinomas, small cellcarcinomas), breast cancers (e.g., inferior ductal carcinomas, inferiorlobular carcinomas, tubular carcinomas), prostate cancers (e.g.,adenocarcinomas), and benign prostatic hyperplasias all show stronglyelevated gli-1 expression levels in certain cases. Accordingly, gli-1expression levels are a powerful diagnostic device to determine which ofthese tissues should be treated with a hedgehog antagonist. In addition,there is substantial correlative evidence that cancers of urothelialcells (e.g., bladder cancer, other urogenital cancers) will also haveelevated gli-1 levels in certain cases. For example, it is known thatloss of heterozygosity on chromosome 9q22 is common in bladder cancers.The ptc-1 gene is located at this position and ptc-1 loss of function isprobably a partial cause of hyperproliferation, as in many other cancertypes. Accordingly, such cancers would also show high gli expression andwould be particularly amenable to treatment with a hedgehog antagonist.

Expression of ptc-1 and ptc-2 is also activated by the hedgehogsignaling pathway, but these genes are inferior to the gli genes asmarkers of hedgehog pathway activation. In certain tissues only one ofptc-1 or ptc-2 is expressed although the hedgehog pathway is highlyactive. For example, in testicular development, Indian hedgehog plays animportant role and the hedgehog pathway is activated, but only ptc-2 isexpressed. Accordingly, these genes are individually unreliable asmarkers for hedgehog pathway activation, although simultaneousmeasurement of both genes are contemplated as a useful indicator fortissues to be treated with a hedgehog antagonist.

Ailments which may be treated by the subject method are disordersspecific to non-humans, such as mange.

In still another embodiment, the subject method can be used in thetreatment of human cancers, particularly basal cell carcinomas and othertumors of epithelial tissues such as the skin. For example, hedgehogantagonists can be employed, in the subject method, as part of atreatment for basal cell nevus syndrome (BCNS), and other humancarcinomas, adenocarcinomas, sarcomas and the like.

In a preferred embodiment, the subject method is used as part of atreatment of prophylaxis regimen for treating (or preventing) basal cellcarcinoma. The deregulation of the hedgehog signaling pathway may be ageneral feature of basal cell carcinomas caused by ptc mutations.Consistent overexpression of human ptc mRNA has been described in tumorsof familial and sporadic BCCs, determined by in situ hybridization.Mutations that inactivate ptc may be expected to result inoverexpression of mutant Ptc, because ptc displays negativeautoregulation. Prior research demonstrates that overexpression ofhedgehog proteins can also lead to tumorigenesis. That sonic hedgehog(Shh) has a role in tumorigenesis in the mouse has been suggested byresearch in which transgenic mice overexpressing Shh in the skindeveloped features of BCNS, including multiple BCC-like epidermalproliferations over the entire skin surface, after only a few days ofskin development, A mutation in the Shh human gene from a BCC was alsodescribed; it was suggested that Shh or other Hh genes in humans couldact as dominant oncogenes in humans. Sporadic ptc mutations have alsobeen observed in BCCs from otherwise normal individuals, some of whichare UV-signature mutations. In one recent study of sporadic BCCs, five.UV-signature type mutations, either CT or COT changes, were found out offifteen tumors determined to contain ptc mutations. Another recentanalysis of sporadic ptc mutations in BCCs and neuroectodermal tumorsrevealed one CT change in one of three ptc mutations found in the BCCs.See, for example, Goodrich et al, (1997) Science 277:1109-13 Xie et al.(1997) Cancer Res 57:2369-72; Oro et al, (1997) Science 276:81.7-21; Xieet al. (1997) Genes Chromosomes Cancer 18:305-9; Stone et al, (1996)Nature 384:129-34; and Johnson et al, (1996) Science 272:1668-71.

The subject method can also be used to treatment patients with BCNS,e.g., to prevent BCC or other effects of the disease which may be theresult of ptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function. Basal cell nevus syndrome is a rare autosomal dominantdisorder characterized by multiple BCCs that appear at a young age. BCNSpatients are very susceptible to the development of these tumors; in thesecond decade of life, large numbers appear, mainly on sun-exposed areasof the skin. This disease also causes a number of developmentalabnormalities, including rib, head and face alterations, and sometimespolydactyly, syndactyly, and spina bifida. They also develop a number oftumor types in addition to BCCs: fibromas of the ovaries and heart,cysts of the skin and jaws, and in the central nervous system,medulloblastomas and meningiomas. The subject method can be used toprevent or treat such tumor types in BENS and non-BCNS patients. Studiesof BCNS patients show that they have both genomic and sporadic mutationsin the ptc gene, suggesting that these mutations are the ultimate causeof this disease.

In another aspect, the present invention provides pharmaceuticalpreparations comprising hedgehog antagonists. The hedgehog antagonistsfor use in the subject method may be conveniently formulated foradministration with a biologically acceptable medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Theoptimum concentration of the active ingredient(s) in the chosen mediumcan be determined empirically, according to procedures well known tomedicinal chemists. As used herein, “biologically acceptable medium”includes an and all solvents, dispersion media, and the like which maybe appropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activity of thehedgehog antagonist, its use in the pharmaceutical preparation of theinvention is contemplated. Suitable vehicles and their formulationinclusive of other proteins are described, for example, in the bookRemingtons Pharmaceutical Sciences (Remington's Pharmaceutical Sciences.Mack Publishing Company, Easton, Pa., USA 1985). These vehicles includeinjectable “deposit formulations”.

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g., pharmaceutical preparations of thehedgehog antagonists suitable for veterinary uses, e.g., for thetreatment of livestock or domestic animals, e.g., dogs.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacions biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a hedgehog antagonist at aparticular target site.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, controlled release patch, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories. Oral and topicaladministrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular hedgehog antagonist employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable and/or sterile carriers andcan also be administered in conjunction with other antimicrobial agentssuch as penicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

V. Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The hedgehog antagonistsaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine. In certainembodiments, the compound included in the pharmaceutical preparation maybe active itself, or may be a prodrug, e.g., capable of being convertedto an active compound in a physiological setting.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam. However, in certain embodiments the subject compounds may besimply dissolved or suspended in sterile water. In certain embodiments,the pharmaceutical preparation is non-pyrogenic, i.e., does not elevatethe body temperature of a patient.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect by overcoming a ptc loss-of-function, hedgehoggain-of-function, or smoothened gain of-function in at least asub-population of cells in an animal and thereby blocking the biologicalconsequences of that pathway in the treated cells, at a reasonablebenefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject antagonistsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present hedgehogantagonists may contain a basic functional group, such as amino oralkylamino, and are, thus, capable of forming pharmaceuticallyacceptable salts with pharmaceutically acceptable acids. The term“pharmaceutically acceptable salts” in this respect, refers to therelatively non-toxic, inorganic and organic acid addition salts ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of the compounds of theinvention, or by separately reacting a purified compound of theinvention in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, for example, Berge et al,(1977) “Pharmaceutical Salts”, J. Pharm Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al. supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) hinders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite day; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

It is known that sterols, such as cholesterol, will form complexes withcyclodextrins. Thus, in preferred embodiments, where the inhibitor is asteroidal alkaloid, it may be formulated with cyclodextrins, such as α-,β- and γ-cyclodextrin, dimethyl-β cyclodextrin and2-hydroxypropyl-β-cyclodextrin.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active hedgehog antagonist.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the hedgehog antagonistsin the proper medium. Absorption enhancers can also be used to increasethe flux of the hedgehog antagonists across the skin. The rate of suchflux can be controlled by either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed preinixes and complete rations can be prepared andadministered are described in reference books (such as “Applied AnimalNutrition”, W.H. Freedman and CO., San Francisco, U.S.A., 1969 or“Livestock Feeds and Feeding” 0 and B books, Corvallis, Ore., U.S.A.,1977),

VI. Synthetic Schemes and Identification of Active Antagonists

The subjects steroidal alkaloids, and congeners thereof, can be preparedreadily by employing the cross-coupling technologies of Suzuki, Stille,and the like. These coupling reactions are carried out under relativelymild conditions and tolerate a wide range of “spectator” functionality.

a. Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g. a variegated library of compounds representedabove, can be screened rapidly in high throughput assays in order toidentify potential hedgehog antagonist lead compounds, as well as torefine the specificity, toxicity, and/or cytotoxic-kinetic profile of alead compound. For instance, ptc, hedgehog, or smoothened bioactivityassays, such as may be developed using cells with either a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function, can be used to screen a library of the subjectcompounds for those having agonist activity toward ptc or antagonistactivity towards hedgehog or smoothened.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds that maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes that need to be carried out. Screeningfor the appropriate physical properties can be done by conventionalmethods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjecthedgehog antagonists. See, for example, Blondelle et al. (1995) TrendsAnal. Chem. 14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899:the Ellman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO94/08051; the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen et al.(1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publicationsWO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCTpublication WO93/20242). Accordingly, a variety of libraries on theorder of about 100 to 1,000,000 or more diversomers of the subjecthedgehog antagonists can be synthesized and screened for particularactivity or property.

In an exemplary embodiment, a library of candidate hedgehog antagonistsdiversomers can be synthesized utilizing a scheme adapted to thetechniques described in the Still et al. PCT publication WO 94/08051,e.g., being linked to a polymer bead by a hydrolyzable or photolyzablegroup, optionally located at one of the positions of the candidateantagonists or a substituent of a synthetic intermediate. According tothe Still et al. technique, the library is synthesized on a set ofbeads, each bead including a set of tags identifying the particulardiversomer on that bead. The bead library can then be “plated” with ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function cells for which an hedgehog antagonist is sought. Thediversomers can be released from the bead, e.g. by hydrolysis.

The structures of the compounds useful in the present invention lendthemselves readily to efficient synthesis. The nature of the structures,as generally described by formulas I to II, allows the combinatorialassembly of such compounds using some combination of Ar, R₁, X, Y, and Zmoieties, as set forth above. For example, these subunits can beattached to the core ring through common acylation or alkylationreactions. The vast majority of such reactions, including those depictedin FIGS. 11, 12, 15, and 16 are both extremely mild and extremelyreliable, and are thus perfectly suited for combinatorial chemistry.Such combinatorial approaches may employ variations on one of the routesdisclosed below.

Many variations on the above and related pathways permit the synthesisof widely diverse libraries of compounds that may be tested asinhibitors of hedgehog function.

Preparation of Exemplary Compounds of the Present Invention

a. Illustrative Synthetic Schemes

Exemplary synthesis schemes for generating hedgehog antagonists usefulin the methods and compositions of the present invention are shown inFIGS. 1-31.

The reaction conditions in the illustrated schemes of FIG. 1-31 are asfollows:

1) R₁CH₂CN, NaNH₂, toluene

(Arzneim-Forsch, 1990, 40, 11, 1242)

2) H₂SO₄, H₂O, reflux

(Arzneim-Forsch, 1990, 40, 11, 1242)

3) H₂SO₄, EtOH, reflux

(Arzneim-Forsch, 1990, 40, 11, 1242)

4) NaOH, EtOH, reflux

5) (Boc)₂O, 2M NaOH, THF 6) Lit R₁X, THF

(Merck Patent Applic # WO 96/06609)

7) Pd—C, H₂, MeOH

8) t-BuONO, CuBr, HBr, H₂O

(J. Org, Chem. 1977, 42, 2426)

9) ArB(OH)₂, Pd(PPh₃)₄, Dioxane

(J. Med. Chem., 1996, 39, 217-223)

10) R₁₂(H)C═CR₁₃R₁₄, Pd(OAc)₂, Et₃N, DMF

(Org. React. 1982, 27, 345)

11) Tf₂O, THF

(J, Am. Chem. Soc. 1987, 109, 5478-5486)

12) ArSnBu₃, Pd(PPh₃)₄, Dioxane

(J. Am. Chem., Soc. 1987, 109, 5478-5486)

13) KMnO₄, P, H₂O

(J. Med. Chem. 1996, 39, 217-223)

14) NaOR₁, THF 15) NaSR₁, THF 16) HNR₁R₁₃, THF 17) HONO, NaBF₄

(Adv. Fluorine Chem. 1965, 4, 1-30)

18) Pd(OAC)₂, NaH, DPPF, PhCH₃, R₁OH

(J. Org. Chem. 1997, 62, 5413-5418)

19) i. R₁X, Et₃N, CR₂Cl₂, ii. R₁₃X

20) SOCl₂, cat DMF 21) CH₂N₂, Et₂O 22) Ag₂O, Na₂CO₃, Na₂S₂O₃, H₂O

(Tetrahedron Lett. 1979, 2667)

23) AgO₂CPh, Et₃N, MeOH

(Org. Syn., 1970, 50, 77; J. Am. Chem. Soc, 1987, 109, 5432)

24) LiOH, THF-MeOH 25) (EtO)₂P(O)CH₂CO₂R, BuLi, THF

26) MeO₂CCH(Br)═P(Ph)₃, benzene

27) KOH or KOtBu

28) Base, X(CH₂)_(n)CO₂R29) DPPA, Et₃N, toluene

(Synthesis 1985, 220)

30) HONO, H₂O 31) SO₂, CuCl, HCl, H₂O

(Synthesis 1969, 1-10, 6)

32) Lawesson's reagent, toluene

(Tetrahedron Asym. 1996, 7, 12, 3553)

33) R₂M, solvent34) 30% H₂O₂, glacial CH₃CO₂H

(Helv. Chim, Acta. 1968, 349, 323)

35) triphosgene, CH₂Cl₂

(Tetrahedron Lett., 1996, 37, 8589)

36) i. (EtO)₂P(O)CHLiSO₂Oi-Pr, THF, ii. NaI

37) Ph₃PCH₃I, NaCH₂S(O)CH₃, DMSO

(Synthesis 1987, 498)

38) Br₂, CHCl₃ or other solvent

(Synthesis 1987, 498)

39) BuLi, Bu₃SnCl₄ 40) ClSO₂OTMS, CCl₄

(Chem. Ber, 1995, 128, 575-580)

41) MeOH—HCl, reflux

42) LAH, Et₂O or LiBH₄, EtOH or BH₃-THF

(Tetrahedron Lett., 1996, 37, 8589)

43) MsCl, Et₃N, CH₂Cl₂

(Tetrahedron Lett., 1996, 37, 8589)

44) Na₂SO₃, H₂O

(Tetrahedron Lett., 1996, 37, 8589)

45) R₂R₄NH, Et₃N, CH₂Cl₂

46) R₂M, solvent

47) CH₃NH(OCH₃), EDC, HOBt, DIEA, CH₂Cl₂ or DMF

(Tetrahedron Lett, 1981, 22, 3815)

48) MeLi, THF

49) mCPBA, CH₂Cl₂

50) HOBO, Cu₂O, Cu(NO₃)₂, H₂O

(J. Org. Chem. 1977, 42, 2053)

51) R₁M, solvent

52) HONO, NaS(S)COEt, H₂O

(Org. Synth, 1.947, 27, 81)

53) HSR₂ or HSR₄, CH₂Cl₂

54) i-BuOC(O)Cl, Et₃N, NH₃, THF

55) R₂R₄NH, CH₂Cl₂, NaBH(OAc)₃ 56) R₂R₄NH, MeOH/CH₃CO₂H, NaBH₃CN 57)R₂OH, EDC, HOBt, DIEA, CH₂Cl₂ or DMF 58) R₂OH, HBTU, HOBt, DIEA, CH₂Cl₂or DMF 59) R₂R₄NH, EDC, HOBt, DIEA, CH₂Cl₂ or DMF 60) R₂R₄NH, HOBt,DIEA, CH₂Cl₂ or DMF 61) POCl₃, Py, CH₂Cl₂

62) R₂R₄NCO, solvent63) R₂OC(O)Cl, Et₃N, solvent

64) R₂CO₂H, EDC or HBTU, HOBt, DIEA, CH₂Cl₂ or DMF

65) R₂X, Et₃N, solvent

66) (CH₃S)₂C═N(CN), DMF, EtOH

(J. Med, Chem, 1994, 37, 57-66)

67) R₂SO₂Cl, Et₃N, CH₂Cl₂ 68) R₂- or R₃- or R₄CHO, MeOH/CH₃CWH, NaBH₃CN

(Synthesis 1975, 135-146)

69) Boc(Tr)-D or L-CysOH, FIBTU, HOBt, DIEA, CH₂Cl₂ or DMF

70) Boc(Tr)-D ort, CysH, NaBH₃CN, MeOH/CH₃CO₂H

(Synthesis 1975, 135-146)

71) S-Tr-N-Boe cysteinal, ClCH₂CH₂Cl or THF, NaBH(OAc)₃

(J. Org. Chem., 1996, 61, 3849-3862)

72) TFA, CH₂Cl₂, Et₃SiH or (3:1:1) thioanisole/ethanedithiol/DMS

73) TFA, CH₂Cl₂

74) DPPA, Et₃N, toluene, HOCH₂CH₂SiCH₃

(Tetrahedron Lett. 1984, 25, 3515)

75) TBAF, THF 76) Base, TrSH or BnSH 77) Base, R₂X or R₄X 78) R₃NH₂,MeOH/CH₃CO₂H, NaBH₃CN 79) N₂H₄, KOH

80) Pd2(dba)₃, P(o-tol)₃, RNH₂, NaOtBit, Dioxane, R₁NH₂

(Tetrahedron Lett. 1996, 37, 7181-7184).

81) Cyanamide,

82) Fmgc-Cl, sodium bicarbonate.83) BnCOCl, sodium carbonate,84) AllylOCOCl, pyridine.85) Benzyl bromide, base.86) Oxalyl chloride, DMSO.

87) RCONH₂.

88) Carbonyldiimidazoie, neutral solvents (e.g., DCM, DMF, Tiff,toluene).89) Thiocarbonyldiimidazole, neutral solvents (e.g., DCM DMF, THF,toluene).90) Cyanogen bromide, neutral solvents (e.g., DCM, DMF, THF, toluene).

91) RCOCl, Triethylamine 92) RNHNH₂, EDC. 93) RO₂CCOCl, Et₃N, DCM.

94) MsOH, Pyridine (J. Het. Chem., 1980, 607.)95) Base, neutral solvents (e.g., DCM, toluene, THF),

96) H₂NOR, EDC. 97) RCSNH₂.

98) RCOCHBrR, neutral solvents (e.g., DCM, DMF, THF, toluene), (Org,Proc. Prep, Intl., 1992, 24, 127).

99) CH₂N₂, HCl. (Synthesis, 1993, 197).

100) NH2NHR, neutral solvents (e.g., DCM, DMF, THF, toluene),

101) RSO₂Cl, DMAP. (Tetrahedron Lett, 1993, 34, 2749).

102) Et₃N, RX. (J. Org. Chem., 1990, 55, 6037).103) NOCl or Cl₂ (J. Org. Chem., 1990, 55, 3916),104) H₂NOH, neutral solvents (e.g., DCM, DMF, THF, toluene).105) RCCR, neutral solvents (DCM, THF, Toluene).106) RCHCHR, neutral solvents (DCM, THF, Toluene).

107) H₂NOH, HCL

108) Thiocarbonyldiimidazole, SiO₂ or BF₃OEt₂). (J. Med. Chem., 1996,39, 5228).109) Thiocarbonyldiimidazole, DBU or DBN, (J, Med. Chem., 1996, 39,522$).

110) HNO₂, HCl.

111) ClCH₂CO₂Et (Org. Reactions, 1959, 1.0, 143),112) Morpholine enamine (Eur. J. Med. Chem., 1982, 17, 27).

113) RCOCHR′CN 114) RCOCHR′CO₂Et 115) Na₂SO₃ 116) H₂NCHRCO₂Et 117)EtO2CCHRNCO 118) RCNHNH₂.

119) RCOCO₂H, (J. Med. Chem., 1995, 38, 3741).

120) RCHO, KOAc. 121) 2-Fluoronitrobenzene. 122) SnCl₂, EtOH, DMF. 123)RCHO, NaBH₃CN, HOAc. 124) NH₃, MeOH. 125) 2,4,6-Me₃PhSO₂NH₂, 126) Et₂NH,CH₂Cl₂ 127) MeOC(O)Cl, Et₃N, CH₂Cl₂ 128) R₂NH₂, EDC, HOBT, Et₃N, CH₂Cl₂129) DBU, PhCH₃ 130) BocNHCH(CH₂STr)CH₂NH₂, EDC, HOBT, Et₃N CH₂Cl₂ 131)R₂NHCH₂CO₂Me, HBTU, HOBT, Et₃N, CH₂Cl₂ 132) BocNHCH(CH₂STr)CH₂OMs,LiHMDS, THF 133) R₂NHCH₂CO₂Me, NaBH(OAc)₃, ClCH₂CH₂Cl or THF 134)R₂NHCH₂CH(OEt)₂, HBTU, HOBT, Et₃N, CH₂Cl₂ 135) NaBH(OAc)₃, ClCH₂CH₂Cl orTHF, AeOH. 136) Piperidine, DMF. 137) Pd(Ph₃P)₄, Bu₃SnH. 138) RCO₂H,EDC, HOBT, Et₃N, DCM.

139) RNH₂, neutral solvents.

140) RCHO NaBH₃CN, HOAc.

141) RNCO, solvent.

142) RCO₂H, EDC or HBTU, HOBt, DIEA, CH₂Cl₂ or DMF. 143) RCOCl,Triethylamine 144) RSO₂Cl, Et₃N, CH₂Cl₂. 145) SnCl₂, EtOH, DMF. 146)RNH₂, EDC, HOBt, DIEA, CH₂Cl₂, or DMF. 147) Dibromoethane, Et₃N, CH₂Cl₂

148) Oxalyl chloride, neutral solvents.

149) LiOH, THF-MeOH.

150) Carbonyldiimidazole, neutral solvents (e.g., DCM, DMF, TIMtoluene).

151) RNH₂, Et₃N, CH₂Cl₂. 152) Base, RX. 153) DBU, PhCH₃

154) DPPA, Et₃N, toluene (Synthesis 1985, 220)

155) SOCl₂, cat DMF, 156) ArH, Lewis Acid (AlCl₃, SnCl₄, TiCl₄), CH₂Cl₂.

157) H₂NCHRCO₂Et, neutral solvents.

158) BocHNCHRCO₂H, EDC OR HBTU, HOBt, DIFA, CH₂Cl₂ or DMF, 159) TFA,CH₂Cl₂. b Screening Assays

There are a variety of assays available for determining the ability of acompound to agonize ptc function or antagonize smoothened or hedgehogfunction, many of which can be disposed in high-throughput formats. Inmany drug-screening programs that test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Thus, libraries of synthetic and natural products can be sampled forother compounds that are hedgehog antagonists.

In addition to cell-free assays, test compounds can also be tested incell-based assays. In one embodiment, cell which have a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function phenotype can be contacted with a test agent ofinterest, with the assay scoring for, e.g., inhibition of proliferationof the cell in the presence of the test agent.

A number of gene products have been implicated in patched-mediatedsignal transduction, including patched, transcription factors of thecubitus interruptus (ci) family, the serine/threonine kinase fised (fu)and the gene products of costal-2, smoothened and suppressor of fused.

The induction of cells by hedgehog proteins sets in motion a cascadeinvolving the activation and inhibition of downstream effectors, theultimate consequence of Which is, in some instances, a detectable changein the transcription or translation of a gene. Potential transcriptionaltargets of hedgehog-mediated signaling are the patched gene (Hidalgo andIngham, 1990 Development 110, 291-301; Marigo et al., 1996) and thevertebrate homologs of the drosophila cubitus interruptus gene, the GLIgenes (Hui et al. (1994) Dev Biol 162:402.413). Patched gene expressionhas been shown to be induced in cells of the limb bud and the neuralplate that are responsive to Shh. (Marigo et al. (1996) PNAS 93:9346-51;Marigo et al. (1996) Development 122:12.25-1233). The Gli genes encodeputative transcription factors having zinc finger DNA binding domains(Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzler et al. (1990) MolCell Biol 10:634-642). Transcription of the Gli gene has been reportedto be upregulated in response to hedgehog in limb buds, whiletranscription of the Gli3 gene is downregulated in response to hedgehoginduction (Mango et al. (1996) Development 122:1225-1233). By selectingtranscriptional regulatory sequences from such target genes, e.g., frompatched or Gli genes, that are responsible for the up- ordown-regulation of these genes in response to hedgehog signalling, andoperatively linking such promoters to a reporter gene, one can derive atranscription based assay which is sensitive to the ability of aspecific test compound to modify hedgehog-mediated signalling pathways.Expression of the reporter gene, thus, provides a valuable screeningtool for the development of compounds that act as antagonists ofhedgehog.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on ptc loss-of-function, hedgehoggain-of-function, smoothened gain-of-function, or stimulation by SHHitself. The amount of transcription from the reporter gene may bemeasured using any method known to those of skill in the art to besuitable. For example, mRNA expression from the reporter gene may bedetected using RNAse protection or RNA-based PCR, or the protein productof the reporter gene may be identified by a characteristic stain or anintrinsic biological activity. The amount of expression from thereporter gene is then compared to the amount of expression in either thesame cell in the absence of the test compound or it may be compared withthe amount of transcription in a substantially identical cell that lacksthe target receptor protein. Any statistically or otherwise significantdecrease in the amount of transcription indicates that the test compoundhas in some manner agonized the normal ptc signal (or antagonized thegain-of-function hedgehog or smoothened signal), e.g., the test compoundis a potential hedgehog antagonist.

VII. Business Methods

One aspect of the present invention relates to a kit comprising ahedgehog antagonist, e.g., as described herein, for treating orpreventing basal cell carcinoma in a patient, preferably a human, and inassociation with instructions (written and/or pictorial) describing theuse of the formulation for treatment or prevention of basal cellcarcinoma, and, optionally, warnings of possible side effects anddrug-drug or drug-food interactions.

Another aspect of the present invention relates to a kit comprising ahedgehog antagonist, e.g., as described herein, wherein the hedgehogantagonist is administered as part of a therapeutic or cosmeticapplication, such as those described above (e.g., regulation of neuraltissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primative gut, regulation ofhematopoietic function, and regulation of skin and hair growth), to apatient, preferably a human, in association with instructions (writtenand/or pictorial) describing the therapeutic or cosmetic application,and optionally, warnings of possible side effects and drug-drug ordrug-food interactions.

The invention further contemplates a method for conducting apharmaceutical business, comprising: (a) manufacturing a pharmaceuticalpreparation comprising a sterile pharmaceutical excipient and a hedgehogantagonist; and (b) marketing (e.g., providing promotional and/orinformative presentations (such as displays, telemarketing, andlectures), products (such as trial samples of the preparation), and/ordocumentation (including leaflets, pamphlets, websites, posters, etc.))to healthcare providers, such as doctors, hospitals, clinics, etc., abenefit of using the pharmaceutical preparation for treating orpreventing basal cell carcinoma.

Another aspect of the present invention relates to a method forconducting a pharmaceutical business, comprising: (a) manufacturing apharmaceutical preparation comprising a sterile pharmaceutical excipientand a hedgehog antagonist; and (b) marketing (e.g., providingpromotional and/or informative presentations (such as displays,demonstrations, telemarketing, and lectures), products (such as trialsamples of the preparation), and/or documentation (including leaflets,pamphlets, websites, posters, etc.)) to healthcare providers, such asdoctors, hospitals, clinics, etc., a benefit of using the pharmaceuticalpreparation as part of a therapeutic or cosmetic application, such asregulation of neural tissues, bone and cartilage formation and repair,regulation of spermatogenesis, regulation of smooth muscle, regulationof lung, liver and other organs arising from the primitive gut,regulation of hematopoietic function, and regulation of skin and hairgrowth.

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) providing a distributionnetwork for selling the pharmaceutical composition comprising a sterilepharmaceutical excipient and a hedgehog antagonist; and (b) providinginstruction material to patients or physicians for using thepharmaceutical composition for treating or preventing basal cellcarcinoma.

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) providing a distributionnetwork for selling the pharmaceutical composition comprising a sterilepharmaceutical excipient and a hedgehog antagonist; and (b) providinginstruction material to patients or physicians for using thepharmaceutical composition as part of a therapeutic or cosmeticapplication, such as those described above (e.g., regulation of neuraltissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primitive gut, regulation ofhematopoietic function, and regulation of skin and hair growth).

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) determining an appropriatepharmaceutical preparation and dosage of a hedgehog antagonist fortreatment or prevention of basal cell carcinoma; (b) conductingtherapeutic profiling of the pharmaceutical composition for efficacy andtoxicity in animals; (c) providing a distribution network for selling apharmaceutical composition having an acceptable therapeutic profile;and, optionally, (d) providing a sales group for marketing thepreparation to healthcare providers.

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) determining an appropriatepharmaceutical preparation and dosage of a hedgehog antagonist fortreatment or prevention of a medical or cosmetic condition describedabove; (b) conducting therapeutic profiling of the pharmaceuticalcomposition for efficacy and toxicity in animals in treating orpreventing the condition; (c) providing a distribution network forselling a pharmaceutical composition having an acceptable therapeuticprofile for treating or preventing the condition; and, optionally, (d)providing a sales group for marketing the preparation to healthcareproviders for the treatment or prevention of the condition.

Yet another aspect of the invention provides a method for conducting apharmaceutical business, comprising: (a) determining an appropriatepharmaceutical composition and dosage of a hedgehog antagonist fortreatment or prevention of basal cell carcinoma; and (b) licensing, to athird party, rights for further development and sale of thepharmaceutical composition.

Yet another aspect of the invention provides a method for conducting apharmaceutical business, comprising: (a) determining an appropriatepharmaceutical composition and dosage of a hedgehog antagonist fortreatment or prevention of a medical or cosmetic condition as describedabove; and (b) licensing, to a third party, rights for furtherdevelopment and sale of the pharmaceutical composition for the treatmentor prevention of the condition.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Experimental Synthesis of Exemplary Inhibitors

Route 1 General Methods.

Unless otherwise noted, all reactions were conducted in oven-driedglassware using HPLC grade solvents. Purification of compounds werecarried out using silica gel 40-63u 60A deactivated with 5% Et₃N (FlashColumn Chromatography) or Jones Chromatography Flash Master 11.Thin-layer chromatography (TLC) was done on plates Kieselgel 60 F₂₅₄(Merck). ¹H-NMR was measured with a Bruker 300 NMR spectrometer indeuteraied-solvents as the internal reference. Low-resolution massspectral data were obtained on a Micromass platform LCZ. Hewlett-Packard1100 was used to determine the purity (LC spectra).

2-Chloro-5-nitro-N-(2-nitro-phenyl)benzamide 1

To a solution of 2-nitro-phenylamine (15.00 g, 109 mmol) in CH₂Cl₂ (90ml), was added pyridine (43 ml, 2.86 vols) at rt followed by dropwiseaddition of 2-chloro-4-nitro-benzoyl chloride (2740 g, 125 mmol)dissolved in CH₂Cl₂ (100 ml) at 0° C. over 45 minutes. The reactionmixture was stirred for 3 h at rt. Once the reaction had gone tocompletion (monitored by LCMS), the crude mixture was evaporated todryness. The crude brown residue obtained was slurried in ethyl acetateand filtered to give the title compound (33 g, 94%) as a yellow solid:¹H NMR (CDCl₃) δ 10.91 (s, 1H), 8.85 (dd, 1H), 8.53 (app d, 1H),8.28-8.21 (m, 2H), 7.70 (dd, 7.64 (d, 1H), 7.25 (dd, 1H); MS (ES,positive mode): 363 [M⁺+1+41] and 322 [M⁺+1].

3-(1H-Benzoimidazol-2-yl)-4-chloro-phenylamine 2

Iron powder (15.42 g, 280 mmol) was added to a solution of amide 1(15.00 g, 46.7 mmol) in toluene (125 ml) and acetic acid (60 nil). Themixture was heated at 125° C. for about 1 h (completion of the reactionwas monitored by LCMS) and the resulting crude mixture was filteredthrough a cotton wool plug (to remove iron particles). The crudesolution was evaporated to dryness, diluted with EtOAc, washed withsaturated NaHCO₃ then brine, dried over MgSO₄, filtered, andconcentrated at reduced pressure. The resulting brown solid wasredissolved in CH₂Cl₂ and few drops of hexane were added to form a solidwhich was isolated by filtration to give 2 (5.66 g, 42%) as a beigesolid: ¹H NMR (CD₃OD) δ 7.64 (broad s, 2H), 7.30-7.26 (m, 3H), 7.13 (appd, 1H), 6.84 (dd, 1H); MS (ES, positive mode): 244 [M⁺+1].

Synthesis of Urea 3

Pyridine (0.12 ml, 1.41 mmol) was added to a solution of amine 2 (300mg, 1.23 mmol) in CH₂Cl₂ (3.0 ml). A solution of 3,5-dimethoxyphenylisocyanate (253 mg, 1.41 mmol) in CH₂Cl₂ (4.0 ml) was added dropwise at0° C. and left stirring for 3 h at rt (completion of the reaction wasmonitored by LCMS). A 1M solution of citric acid was added to quench thereaction, the organic phase was separated and evaporated to dryness. Thecrude mixture was diluted with EtOAc, washed with saturated NaHCO₃ thenbrine, dried over MgSO₄, filtered and concentrated under reducedpressure. The resulting crude product was purified by flashchromatography (EtOAc:hexane, gradient elution) to afford 3 (40 mg, 8%):¹H NMR (CD₃OD) δ 8.05 (app d, 1H), 7.82 (d, 1H), 7.81 (d, 1H) 7.80(broad s, 1H), 7.67 (d, 1H), 7.45 (broad m, 2H), 6.83 (s, 1H), 6.82 (s,1H), 6.33 (broad m, 1H), 191 (s, 6H); MS (ES, positive mode): 423[M⁺+1]; LC (1.22, purity>97%).

Synthesis of Amide 4

A solution of 3,4,5-trimethoxy benzoyl chloride (272 mg, 1.18 mmol) inCH₂Cl₂ (4.0 ml) was added to a solution of amine 2 (250 mg, 1.03 mmol)in CH₂Cl₂ (3.0 ml) at 0° C. under nitrogen. The reaction mixture leftstirring overnight at rt (completion of the reaction was monitored byLCMS). The crude mixture was diluted with EtOAc containing approximately4% MeOH, washed with saturated NaHCO₃, brine, dried over MgSO₄, filteredand concentrated under reduced pressure. The resulting crude product waspurified by preparative LC to give 4 (25 mg, 6%) as a colorless solid:¹H NMR (CD₃OD) δ 8.31 (app d, 1H), 7.98 (dd, 1H), 7.76-7.74 (m, 2H),7.67 (d, 1H), 7.46-7.44 (m, 2H), 7.34 (broad s, 2H); MS (ES, positivemode): 438 [M⁺+1]; LC (1.15, purity>95%).

Synthesis of Thiourea 5

Pyridine (0.2 ml, 2.36 mmol) was added to a solution of amine 2 (250 mg,1.03 mmol) in CH₂Cl₂ (3.0 ml) at 0° C. followed by dropwise addition ofa solution of benzodioxane isothiocyanate (228 mg, 1.18 mmol) in CH₂Cl₂(4.0 ml). The reaction mixture was left stirring overnight at rt(completion of the reaction was monitored by LCMS). A solution of 1 Mcitric acid was added to the crude reaction mixture, and the organicphase was separated and evaporated to dryness. The crude solid wasdiluted with EtOAc, washed successively with saturated NaHCO₃ and brine,and then dried over MgSO₄, filtered and concentrated under reducedpressure. The resulting crude product was purified by flashchromatography (30-50% EtOAc in hexane) to afford 5 (95 mg, 21%) as apale yellow solid: ¹H NMR (CD₃OD) δ 7.71 (app d, 1H), 7.55 (dd, 1H),7.50 (broad s, 2H), 7.40 (d, 1H), 7.19-7.13 (broad m, 214), 6.79 (s,114), 6.67 (s, 214) 4.08 (s, 4H); MS (ES, positive mode): 438 [M⁺+1]; LC(0.941, purity>98%).

Synthesis of Sulfonamide 6

Pyridine (0.20 ml, 2.36 mmol) was added to a solution of amine 2 (250mg, 1.03 mmol) in CH₂Cl₂ (3.0 ml) at 0° C. followed by dropwise additionof phenylsulfonyl chloride (0.15 ml, 1.18 mmol) in CH₂Cl₂ (4.0 ml). Thereaction mixture was stirred for 1 h (completion of the reaction wasmonitored by LCMS). A solution of 1 M citric acid was added to the crudereaction mixture, the organic phase was separated and evaporated todryness. The crude solid was diluted with EtOAc, washed successivelywith saturated NaHCO₃ and brine, dried over MgSO₄, filtered, andconcentrated under reduced pressure. The resulting crude mixture whichcontained mainly bis-alkylated product was purified by flashchromatography (1:39:60, MeOH:EtOAc:Hexane) to give 6 (20 mg, 5%) as acolorless solid: ¹H NMR (CD₃OD) δ 8.01-7.99 (broad d, 2H), 7.97 (d, 1H)7.95 (d, 1H), 7.84 (app d, 1H), 7.76-7.74 (m, 414), 7.68 (d, 1H), 7.66(d, 1H), 7.55 (dd, 1H); MS (ES, positive mode) 384 [M⁺+1]; LC (1.190,purity>99%).

Route 2

2-(2-Methyl-5-nitro-phenyl)-1H-benzimidazole 7

Benzene-1,2-diamine (2.60 g, 25 mmol) and 2-methyl-5-nitrobenzoic acid(5.0 g, 27.6 mmol) were suspended in polyphosphoric acid (30 ml, 11 vol)and heated to 140° C. overnight. Once the reaction had gone tocompletion (monitored by LCMS), the crude green mixture was dissolved inwater, basified with NaOH (pH. 14) and extracted into EtOAc (x 2). Theorganic phase was washed with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure to give 7 (4.70 g, 74%) as anorange/brown solid: ¹H NMR (C₂D₆SO) δ 13.20 (s, 1H), 8.80 (app d, 1H),8.84 (dd, 1H), 7.89-7.87 (m, 2H), 7.74 (d, LH), 7.40-7.45 (m 2H), 2.90(s, 3H); MS (ES, positive mode): 254 [M⁺+1].

3-(1H-Benzimidazol-2-yl)-4-methyl-phenylamine 8

Iron powder (3.0 g, 50.9 mmol) was added to a solution ofnitrobenzimidazole 7 (4.30 g, 16.9 mmol) in toluene (30 ml) and aceticacid (30 ml), and the reaction mixture was heated at 125° C. for about 1h (completion of the reaction was monitored by TLC). The resulting crudemixture was filtered through a cotton wool plug (to remove ironparticles). The crude solution was evaporated to dryness, diluted withEtOAc, washed with saturated NaHCO₃ (until the pH=7), and the aqueousphase was back-extracted with EtOAc. The combined organics were washedwith brine, dried over MgSO₄, filtered and concentrated at reducedpressure to give 8 (2.70 g, 73%) a brown solid: ¹H NMR(C₂D₆SO) δ 12.55(s, 1H), 7.73 (d, 1H), 7.57 (d, 1H), 7.16 (broad m, 2H), 7.09 (d, 1H),7.01 (app d, 1H), 6.70 (dd, 1H) 5.15 (broad s, 2H), 2.39 (s, 3H); MS(ES, positive mode): 224 [M⁺+1].

Synthesis of Amide 9.

Benzimidazolphenylamine 8 (250 mg, 1.12 mmol) and 3-5-dimethoxy benzoylchloride (246 mg, 1.23 mmol) were suspended in CH₂Cl₂ (10.0 ml) andstirred overnight at rt. The crude mixture was diluted with EtOAc, thenwashed with citric acid. The aqueous phase was basified with NaOH andextracted into EtOAc. The combined organic extracts were washed withbrine, dried over MgSO₄, filtered and evaporated under reduced pressureto give 9 as colorless solid: ¹H NMR(C₂D₆SO) δ 12.65 (s, 1H), 10.35 (s,1H), 8.19 (s, 1H), 7.86 (d, 1H), 7.74 (d, 1H), 7.61 (d, 1H), 7.32 (d,1H), 7.42-7.32 (m, 2H), 7.20 (s, 2H), 6.79 (s, 3.89 (s, 6H), 2.61 (s,3H); MS (ES, positive mode): 388 [M⁺+1]; LC (1.19, purity>97%).

Synthesis of Amide 10

Benzimidazolphenylamine 8 (250 mg, 1.12 mmol) andbenzodioxane-4-carbonyl chloride (220 mg, 1.23 mmol) were suspended inCH₂Cl₂ (10.0 ml) and stirred overnight at rt. The crude mixture wasdiluted with EtOAc, then washed with citric acid. The aqueous phase wasbasified with NaOH and extracted into EtOAc. The combined organicextracts were washed with brine, dried over MgSO₄, filtered andevaporated under reduced pressure to give 10 as colorless solid: ¹HNMR(C₂D₆SO) δ 12.55 (s, 1H), 10.49 (s, 1H), 9.07 (app d, 1H), 8.70 (dd,1H), 8.25 (dd, 1H), 8.15 (app d, 1H), 7.72 (dd, 1H), 7.64 (d, 1H),7.52-7.48 (m, 1H), 7.38 (d, 1H), 7.31 (d, 1H), 7.17-6.95 (m, 2H), 3.2$(s, 3H); MS (ES, positive mode): 329 [M⁺+1]; LC (0.858, purity>97%).

Route 3

2-(2-Chloro-5-nitro-phenyl)-1H-benzimidazole-5-carbonitrile 12

The 2-amino-4-cyano-phenyl amine (0.5 g, 3.76 mmol) and2-chloro-4-nitro-benzaldehyde (1.39 g, 7.52 mmol) were dissolvedseparately in a minimum amount of acetic acid and mixed together at rt.The orange solid that formed was filtered, washed with acetic acid andheptane until the filtrate was colourless. The solid isolated at thisstage was shown to be the intermediate 11 (600 mg, 45%): ¹H NMR(C₂D₆SO)δ 8.99 (app d, 1H), 8.91 (s, 1H), 8.28 (dd, 1H), 7.84 (d, 1H), 7.57 (s,1H), 7.35 (dd, 1H), 6.75 (d, 1H), 6.36 (s, 21-1); MS (ES, positivemode): 301 [M⁺+1]. Cyclisation of the Intermediate 11: The resultingbrown solid was suspended in acetic acid and heated at 50° C. for 6 h.The crude mixture was diluted with EtOAc, neutralised with NaHCO₃solution, washed with brine, dried over MgSO₄, and concentrated underreduced pressure to give 12 (200 mg, 99%) as a brown solid: ¹H NMR(C₂D₆SO) δ 13.40 (broad 5, 1H), 8.66 (app d, 114), 8.30 (dd, 114) 8.20(s, 1H), 7.90 (d, 1H), 7.76 (d, 1H) 7.50 (d, 1H); MS (ES, positivemode): 299 [M⁺+1].

Biological Assays Lead Compound Discovery/High-Throughput ScreeningAssay

Compounds to be tested are dissolved in DMSO to a concentration of 10mM, and stored at −20° C. To activate the Hedgehog pathway in the assaycells, an octylated (lipid-modified) form of the N-terminal fragment ofthe Sonic Hedgehog protein (OCT-SHH) is used. This N-terminal SHHfragment is produced bacterially. See, for example, Taylor F R, et al.,Biochemistry 2001, 40, 4359-71.

Compounds may be tested in the “Gli-Luc” assay below, using the cellline 10T1/2(s12), wherein the cells contain a Hedgehog-responsivereporter construct utilizin Luciferase as the reporter gene. In thisway, Hedgehog pathway signaling activity can be measured via the Gli-Lucresponse.

10t1/2(s12) cells are plated in a 96-well micro-titer plate (MTP) at20,000 cells/well in full medium [DMEM with 1.0% FBS]. Then plates areplaced in the incubator for incubation overnight (0/N), at 37° C. and 5%CO₂. After 24 h, the medium is replaced with Luciferase-assay medium(DMEM with 0.5% FBS). Compounds are thawed and diluted in assay mediumat 3:1000 (about 300-fold) resulting in a starting concentration ofabout 0.0003 μM to 30 μM.

Subsequently, 150 μl each sample is added to the first wells (intriplicate). The MTP samples are then diluted at 3-fold dilutions to atotal of seven wells, ultimately resulting in a regiment of sevendilutions in triplicate, for each compound. Next, the protein ligandOCT-SHH is diluted in Luciferase-assay medium and added to each well ata final concentration of 0.3 Plates are then returned to the incubatorfor further incubation O/N, at 37° C. and 5% CO₂. After about 24 h,plates are removed from the incubator and the medium isaspirated/discarded. Wells are washed once with assay buffer [PBS+1 mMMg²⁺ and 1 mM Ca²⁺]. Then 50 μl of assay buffer is added to each well.The Luciferase assay reagent is prepared as described by the vendor(LucLite kit from Packard), and 50 μl is added to each well. Plates areincubated at room temperature (RT) for about 30 minutes after which thesignals are read, again at RT, on a Topcount (Packard).

Similar assays were performed using human cell lines (specifically,human embryonic palatal mesenchyme cells, modified with the Gli-Lucconstruct as described above) in a growth medium of MEM/Sodium Pyruvatew/10% MS, and an assay medium of MEM/Sodium Pyruvate w/0.5% FBS. OCT-SHHwas added to reach a final concentration of 1 μg/ml.

Compounds identified in the above assays are depicted in FIG. 32.

Activities of particular compounds are presented below in Table 1(activities based on results from the human-based assay, except forcompounds E-V):

TABLE 1 Compound IC₅₀ (μM) Compound IC₅₀ (μM) A <0.05 B <0.05 C <0.5 D<0.5 E <5 F <5 G <5 H <5 I <5 J <5 K <5 L <5 M <5 N <1 O <5 P <5 Q <10 R<10 S >10 T <5 U <5 V <5 W <0.01 X <0.05 Y <0.05 Z <0.05 A′ <0.01 B′<0.01 C′ <0.05 D′ <0.05 E′ <0.05 F′ <0.05 G′ <0.01 H′ <0.01 I′ <0.05 J′<0.05 K′ <0.01 L′ <0.05 M′ <0.05 N′ <0.1 O′ <0.1 P′ <0.1 Q′ <0.05 R′<0.05 S′ <0.05 T′ <0.05 U′ <0.05 V′ <0.05 W′ <0.1 X′ <0.1 Y′ <0.5 Z′<0.5 A″ <0.5 B″ <0.5 C″ <0.5 D″ <0.5 E″ <0.5 F″ <0.5 G″ <0.5 H″ <0.5 I″<0.5 J″ <0.05 K″ <0.05 L″ <0.05 M″ <0.05 N″ <0.05 O″ <0.1 P″ <0.5 Q″<0.5 R″ <0.5 S″ <0.1 T″ <0.1 U″ <0.1 V″ <0.5 W″ <0.5 X″ <0.5 Y″ <0.5 Z″<0.5 A′″ <0.5 B′″ <0.5 C′″ <0.5 D′″ <0.5 E′″ <0.5 F′″ <1 G′″ <0.1 H′″<0.5 I′″ <0.5 J′″ <0.5 K′″ <0.01 L′″ <0.5 M′″ <0.5 N′″ <0.01 O′″ <1 P′″<0.5 Q′″ <0.05 R′″ <0.5 S′″ <0.05 T′″ <10 U′″ <0.5 V′″ <0.05 W′″ <0.05X′″ <0.5 Y′″ <0.5 Z′″ <0.5 A″″ <0.5 B″″ <0.05 C″″ <0.05 D″″ <0.05 E″″<0.5 F″″ <0.5 G″″ <0.05 H″″ <0.5 I″″ <0.1 J″″ <0.05 K″″ <5 L″″ <0.05 M″″<0.01 N″″ <0.1 O″″ <0.5 P″″ <0.5 Q″″ <0.5 R″″ <5 S″″ <0.5 T″″ <0.5 U″″<0.05 V″″ <0.05 W″″ <0.5 X″″ <0.5 Y″″ <0.001 Z″″ <0.001 A″″′ <0.05 B″″′<0.05 C″″′ <0.5 D″″′ <0.05 E″″′ <0.05 F″″′ <0.5

Effect of Compounds on Basal Cell Carcinoma (BCC) Samples

BCC Culture:

Curettage specimens of basal cell carcinomas that contain some dermalstroma are cultured in organotypic culture, i.e., exposed to theair/liquid interface. BCC samples are injected with vehicle (0.8%sterile NaCl solution) or compound A, and cultures are assembled on topof a plastic grid and incubated for up to three days (with or withoutcompound) in a medium suitable for the long-term culture of human skin.After 3 days of culture, the samples are processed for routinehistology.

The morphological features characteristic of BCCs, e.g., islands ofundifferentiated basal cells, and in some cases, palisading ofperipheral cells and stromal clefting, are maintained when BCCs arecultured in this system. The GLI-1 gene, a pivotal indicator of hedgehogsignaling, remains active at high levels in untreated cultures, asdetermined following exposure to ³³P-labeled RNA probes.

Quantitative In Situ Hybridization:

Briefly, 7 μm sections of paraformaldehyde-fixed, paraffin-embeddedtissue containing large basal cell islands were cleared, re-hydrated,digested with proteinase K, acetylated and hybridized overnight with³³P-labeled RNA probes. After high stringency post-hybridization washes,slides were exposed to a high-sensitivity PhosphorImager screen in thedark at room temperature for several hours. After developing, the[³³P]-signal was scanned using a Cyclone Scanner (Packard). Individualbasal cell islands were selected and the signal quantified and expressedin DLU (digital light units)/mm² using OptiQuant software. The controlvalues were averaged and set to 100%; the values obtained from compoundA-treated samples were expressed as % of the control. Results aredepicted in FIG. 33.

Caspase 3 Immunohistochemistry.

Programmed cell death or apoptosis is an organized process to eliminateunwanted cells that involves caspase activation, DNA fragmentation,membrane blebbing, cell shrinkage, and phagocytosis. Apoptosis wasanalyzed by immunohistochemistry for active caspase 3, a marker thatdetects an early apoptotic event. Standard histology was performed onthe BCC cultures and slides were de-paraffinized, pre-blocked andincubated with primary antibody, then rinsed and incubated withenzyme-labeled secondary antibody according to the manufacturers'instructions. The specific signal was visualized by enzymatic detectionof the antigen-antibody complex. Slides were cover-slipped and imaged.Results are depicted in FIG. 34, where the left panel is control tissue,and the right panel is in the presence of A.

Effect of Subject Compounds on Other Tumor Cells

Colon Cancer Cells:

20,000 colon cancer cells were mixed with 10,000 10T(S12) cells(described above), and the mixture was added to each well in a 96-wellplate. A subject compound or control vehicle was added to the culture onthe next day. After 72 hrs of incubation, the cells were lysed formeasuring the luciferase activity. Compound A was able to inhibit thelucferase activity, which indirectly reflects activity of the hedgehogsignaling pathway, when comparing to vehicle in this assay. FIG. 35depicts the comparative inhibition of hedgehog-induced activation in S12cells for the antibodies 1E6 and 5E1 and compound A, and the comparativeinhibition of endogenous hedgehog activity in colon cancer cells forthese compositions.

Prostate and Bladder Cancer Cells:

Related experiments employing cell lines PC-3, a hormone refractoryprostate cancer cell line, and RT-4, a urothelial bladder cancer cellline, in the same assay are depicted in FIG. 36.

Effects of Subject Compounds on Other Tissues

The probes and primers depicted in FIG. 37 were used to evaluate theeffect of the subject inhibitors on GLI-1 and GAPDH expression usingRT-PCR and Real-Time PCR (Taqman). HEPM cells were grown in the presenceof Octyl-SHH and subject compounds for 24 hrs. Total RNA was harvestedand used in standard RT-PCR to assess expression levels of the GLI-1 andGAPDH transcripts. FIG. 38 depicts the results of testing compound A,0.003-1.0 μM; compound O, 0.003-1.0 μM; −, negative control (DMSOalone); +, positive control (Octyl-SHH@1 mg/ml, no inhibitor).

Cells with homozygous disruption of patched (ptc-null cells) were grownin the presence of subject compounds for 72-96 hrs. Total RNA washarvested and used in standard RT-PCR to assess expression levels of theGLI-1 and GAPDH transcripts.

FIG. 39 depicts the results of testing compound C, at 1 & 10 μM;compound O, at 1 & 10 μM; DMSO, negative control.

HEPM cells were grown in the presence of Octyl-SHH and subject compoundsfor 24 hrs. Total RNA was harvested and converted to cDNA by standard RTreactions. The cDNA was then used in Real-Time PCR to assess expressionlevels of the GLI-1 and GAPDH transcripts. FIG. 40 depicts the resultsof testing compound A, at 0.001-0.3 μM; compound O, at 0.001-0.3 μM;DMSO(−), negative control; OCT-SHH(+), positive control.

CCD-37 (lung fibroblast) cells were grown in the presence of Octyl-SHHand compound A for 24 hrs. Total RNA was harvested and converted to cDNAby standard RT reactions. The cDNA was then used in Real-Time PCR toassess expression levels of the GLI-1 and GAPDH transcripts. FIG. 41depicts the results of testing compound A, at 0.001-1.0 μM; DMSO(−),negative control; OCT-SHH(+), positive control.

All of the references cited above are hereby incorporated by referenceherein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1.-36. (canceled)
 37. A compound of the formula (II):

wherein, as valence and stability permit, X represents —N(R₈)—; Zrepresents —NH— or a direct bond; Y represents —C(═O)—, —C(═S)— or SO₂;A represents O, or NH—; G represents a phenyl or pyridyl ring fused tothe ring to which it is attached; R₁ represents a substituted orunsubstituted benzene ring, cyclopentyl ring, cyclohexyl ring orheterocyclyl ring selected from substituted or unsubstituted thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,benzodioxane, benzodioxole, phthalazine, naphthyridine, quinoxaline,quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine,acridine, pyrimidine, phenanthroline, phenazine, phenarsazine,phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane,oxazole, piperidine, piperazine, morpholine, lactone, lactam,azetidinone, pyrrolidinone, sultam or sultone rings; R₂ represents from0-4 substituents on the ring to which it is attached, where thesubstituents are selected from halogen, lower alkyl, lower alkenyl,aryl, heteroaryl, carbonyl group, thiocarbonyl, ketone, aldehyde, amino,cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, J-R₈, J-OH, J-loweralkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protected forms of the above,or any two R₂, when occurring more than once in a cyclic or polycyclicstructure, taken together form a 4- to 8-membered cycloalkyl, aryl, orheteroaryl; R₃ represents a substituent on the ring to which it isattached, the substituent being at a position on the ring which is parato where the X group is attached or para to where the bicyclic ringcontaining the A group is attached, and the substituent being selectedfrom halogen, hydroxyl, alkoxy, amino, alkylamino, cyano, nitro,substituted or unsubstituted lower alkyl, and acyl, R₈, independentlyfor each occurrence, represents H, lower alkyl or cycloalkyl; and Jrepresents, independently for each occurrence, a chain having from 0-8units selected from CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl; provided that, whenJ is CK₂, R₁ is not a cyclohexyl ring; and provided that, when R₈ is H,Z is a direct bond and Y is —C(═O)—, R₁ is not a disubstituted pyridinering; or a pharmaceutically acceptable salt thereof.
 38. The compound ofclaim 37, wherein X—Y—Z includes an amide, urea, or sulfonamide.
 39. Thecompound of claim 37, wherein R₁ is optionally substituted with from 1-5substituents.
 40. The compound of claim 37, wherein the substituents onR₁ are selected from nitro, halogen, cyano, lower alkyl, acylamino,alkoxy, alkylamino, a substituted or unsubstituted cycloalkyl,heterocyclyl, aryl, and heteroaryl fused to the aryl or heteroaryl ring.41. The compound of claim 37, wherein R₂ represents from 1-4substituents as defined.
 42. The compound of claim 41, wherein R₂represents from 1-4 substituents selected from halogen, cyano, nitro,alkoxy, amino, acylamino, a substituted or unsubstituted cycloalkyl,heterocyclyl, aryl, or heteroaryl fused to G, and substituted orunsubstituted lower alkyl.
 43. The compound of claim 37, wherein R₁represents a substituted or unsubstituted benzene ring, cyclopentylring, cyclohexyl ring, thiophene ring, furan ring, isobenzofuran ring,pyridine ring, benzodioxane ring, or benzodioxole ring.
 44. The compoundof claim 37, wherein R₁ represents a substituted or unsubstitutedbenzene ring or pyridine ring.
 45. The compound of claim 37, which is acompound selected from the following:

or a pharmaceutically acceptable salt thereof.
 46. A pharmaceuticalcomposition comprising one or more compounds according to claim 37formulated together with one or more pharmaceutically acceptablecarriers and/or diluents.